Lentiviral vectors and methods of use thereof

ABSTRACT

The compositions and methods described herein relate to lentiviral vectors that can be used to generate virions/viruses that exhibit enhanced infectivity with respect to monocyte-derived macrophages (MDM) and dendritic cells (MDDC). Such compositions and methods further relate to production of virions/viruses that can be used as components of vaccines that effectively stimulate innate immune responses. In a particular embodiment, compositions and methods described herein relate to production of virions/viruses that can be used as components of human immunodeficiency-1 (HIV-1) vaccines administered to stimulate innate immune responses to HIV-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) from U.S.Provisional Application Ser. No. 61/587,392, filed Jan. 17, 2012, whichapplication is herein specifically incorporated by reference in itsentirety.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by National Institutes of Health (NIH) grant 5R01 A1067059 and NIHtraining grant 5T32 A1007180. Accordingly, the Government has certainrights in the invention.

FIELD OF INVENTION

The compositions and methods described herein relate to lentiviralvectors that can be used to generate virions that exhibit enhancedinfectivity with respect to monocyte-derived macrophages (MDM) anddendritic cells (MDDC). More particularly, compositions and methodsdescribed herein relate to production of virions that can be used ascomponents of vaccines that effectively stimulate innate immuneresponses. In a particular embodiment, compositions and methodsdescribed herein relate to production of virions that can be used ascomponents of human immunodeficiency-1 (HIV-1) vaccines that effectivelystimulate innate immune responses.

BACKGROUND OF INVENTION

Unlike simple retroviruses, lentiviruses encode a set of accessoryproteins (Nef, Vif, Vpr, Vpu, and Vpx), each of which play a specificrole in virus replication and pathogenesis, much of which are directedat evasion of the host adaptive or innate immune response (28, 33, 35,36, 41, 46). Specifically, Nef downregulates cell surface CD4 moleculesand disrupts antigen presentation, and Vif and Vpu serve to counteractthe host restriction factors, APOBEC3G and BST-2/tetherin, respectively.Although the roles of Vpx and Vpr are less well understood, they arethought to aid in evasion of a yet uncharacterized restriction to virusreplication in macrophages and dendritic cells (3, 7, 10, 18, 20, 22,24, 27, 51). Sharing significant sequence and structural homology, thetwo proteins appear to have arisen from the duplication of a commonancestral precursor gene (44). Both are produced late in the viral lifecycle and localize to the nucleus (5, 12, 31). A Vpr gene is present inthe genome of all known lentiviruses, while Vpx is restricted to HIV-2and the SIV of sooty mangabey (SIVsm) and macaque (SIVmac).Interestingly, SIV of the African green monkey encodes only Vpr, butthis protein has some characteristics of Vpx, sharing the same virionpackaging determinant and effect on virus replication in macrophages (1,9).

A distinguishing feature of Vpr and Vpx is that they are packaged intovirions at significant levels (2, 23, 50). Packaging of Vpr and Vpxoccurs as the virion assembles and is mediated by amino acid motifs inthe carboxy-terminal Gag protein p6, such that deletion of p6 preventsincorporation of both Vpr and Vpx (31, 38, 47). The packaging of Vpr andVpx is also virus-specific (25). HIV-1 will not package SIVmac Vpr orVpx, nor will SIVmac package HIV-1 Vpr. By analyzing viruses withmutated or truncated p6, various groups have identified amino acidmotifs in p6 that mediate Vpr and Vpx incorporation. The motif ⁴¹LXXLF⁴⁴(SEQ ID NO: 58) near the p6 carboxy-terminus was reported by Kondo etal. as the major determinant required for the packaging of Vpr (30).Subsequently, Zhu et al. (53) reported that mutation of that motif didnot prevent Vpr packaging. Instead, in a virus deleted for amino acids35-52, the ¹⁵FRFG¹⁸ motif near the amino-terminus of p6 was required.This discrepancy has not yet been resolved. In SIVmac, the Vpx packagingmotif has also been mapped to the N terminal region of p6, specificallyto a conserved ¹⁷DXAXXLL²³ (SEQ ID NO: 60) motif (1). Packaging ofSIVagm Vpr was also dependent on this motif, drawing a resemblance withVpx.

Vpr and Vpx are important for virus replication and pathogenicity invivo, as demonstrated in the rhesus macaque model, where Vpx-deletedSIVmac₂₃₉ is attenuated and Vpx/Vpr-deleted virus is further impaired(17). In vitro, neither Vpr nor Vpx is required for SIVmac replicationin activated CD4 T cells (3, 18, 19). However, in monocyte-derivedmacrophages (MDM) and dendritic cells (MDDC), both proteins enhanceSIVmac infection, although Vpr has a more modest effect. The presence ofVpx and Vpr in the virion suggests that they play a role early in virusreplication. Vpr and Vpx were initially proposed to aid in nuclearimport of the preintegration complex in infection of nondividing cells,a mechanism consistent with their karyophilic properties and theirpresence in the virion (5, 13). However, this view was challenged by thefinding that HIV-1 deleted for Vpr maintained its ability to infectnondividing cells (48). Recently, a role for Vpx and Vpr incounteracting a restriction factor was suggested by the finding thatboth associated with an E3 ubiquitin ligase composed of damaged DNAbinding protein 1 (DDB1), DDB1 cullen associated factor 1 (DCAF1), andCullin 4A (Cul4A) (6, 21, 26, 39, 40, 42). By analogy with Vif, thisfinding suggested that Vpx and Vpr might act as substrate receptors thatinduce the ubiquitination of a host restriction factor (41, 43, 49).Evidence that Vpx plays a role in counteracting an MDM-specific hostrestriction was provided by somatic cell fusion experiments in whichheterokaryons formed between MDM and COS were found to be nonpermissivefor Vpx-deleted SIVmac (40). Furthermore, domain mapping of Vpxlocalized an activation domain at the amino-terminus that might serve asa binding site for the putative host restriction factor (21).

In studies investigating the mechanism by which Vpx promotes theinfection of MDDC, Goujon et al. found that Vpx could facilitateinfection when introduced into the target cell in trans (18, 20). Forthis, MDDC were exposed to virus-like particles (VLP) that contained Vpxand then were infected with Vpx-deleted SIVmac. The VLP dramaticallyenhanced the infectability of the cells. Additionally, Gramberg et al.found that VLP generated with a codon-optimized Vpx expression vectorcould boost the infection of MDM as much as 100-fold, while Vpr hadlittle effect (21). In these cells, Vpx was found to relieve a block toinfection at early reverse transcription or at uncoating. Interestingly,Vpx also dramatically enhanced HIV-1 infection of MDM and MDDC, eventhough the virus does not itself encode this accessory protein.Recently, Manel et al. used this property to achieve high levels ofHIV-1 infected MDDC (32). The infected MDDC strongly induced innateimmune defenses, resulting in the production of type-I IFN andupregulation of CD86. Surprisingly, the trigger that induced thisresponse was not the in-coming virus but, rather, the newly produced Gagprotein.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

To date, Vpx has been used to enhance HIV-1 infection of MDM and MDDC bypretreating cells with VLP. In this study, the present inventors soughtto investigate the role of Vpx by generating an HIV-1 vector that wouldpackage the protein. Such a vector would obviate the need forVpx-containing VLP. Virions/viruses generated using the resulting vectorwould have an enhanced ability to infect MDDC and MDM and be expected toinduce an innate immune response in the cells. The present inventorsconstructed a vector having the desired properties by introducing afragment of SIVmac₂₃₉ p6 into HIV-1 p6. In a further embodiment, thevector comprising the introduced fragment of SIVmac₂₃₉ p6 into HIV-1 p6was further modified by placing vpx in the Nef position. Moreparticularly, vpx was inserted into the first codon of Nef Thisarrangement prevents Nef expression, because protein synthesis isterminated by the vpx stop codon. Virions generated using the resultingvectors efficiently infected MDM and MDDC and induced high levels oftype I IFN. In a MDDC/T cell transfer assay, the engineered virions soproduced replicated much more efficiently than those generated usingwild-type vector. These findings suggest that an important role of Vpxis to enhance the ability of virus to be transmitted from MDM and MDDCto CD4 T cells. Furthermore, such virions are useful for the productionof an HIV vaccine that stimulates innate immune responses and for thegeneration of lentiviral virions that more efficiently infect these celltypes.

In accordance with the present findings, a chimeric vector comprisingHIV-1 nucleic acid sequences and SIVmac₂₃₉ nucleic acid sequences ispresented herein, wherein the SIVmac₂₃₉ nucleic acid sequences encode anSIVmac₂₃₉ amino acid sequence consisting of a minimal Vpx packagingmotif that confers Vpx packaging activity to the chimeric vector. In anembodiment thereof, the chimeric vector does not comprise any SIVmacnucleic acid sequences except for or in addition to the minimal Vpxpackaging motif.

In a further embodiment, the chimeric vector further comprises vpxinserted in nef of the HIV-1 nucleic acid sequences. In a particularembodiment thereof, the vpx is SIVmac₂₃₉ vpx. In a more particularembodiment, the vpx is a codon-optimized SIVmac₂₃₉ vpx. In a stillfurther embodiment, the chimeric vector that further comprises vpx, doesnot comprise any SIVmac₂₃₉ nucleic acid sequences except for or inaddition to the minimal Vpx packaging motif and the vpx.

In a further aspect, the minimal Vpx packaging motif consists of atleast 10 contiguous amino acids of SIVmac₂₃₉ comprising of¹⁷DPAVDLLKNY²⁶ (SEQ ID NO: 1), wherein the 5′ terminus of the minimalVpx packaging motif is the aspartic acid (D) at amino acid position 17of the SIVmac₂₃₉ amino acid sequence. More particularly, the minimal Vpxpackaging motif consists of

(SEQ ID NO: 1) ¹⁷DPAVDLLKNY²⁶, (SEQ ID NO: 2) ¹⁷DPAVDLLKNYM²⁷,(SEQ ID NO: 3) ¹⁷DPAVDLLKNYMG²⁸, (SEQ ID NO: 4) ¹⁷DPAVDLLKNYMQL²⁹,(SEQ ID NO: 5) ¹⁷DPAVDLLKNYMQLG³⁰, (SEQ ID NO: 6) ¹⁷DPAVDLLKNYMQLGK³¹,(SEQ ID NO: 7) ¹⁷DPAVDLLKNYMQLGKQ³², (SEQ ID NO: 8)¹⁷DPAVDLLKNYMQLGKQQ³³, (SEQ ID NO: 9) ¹⁷DPAVDLLKNYMQLGKQQRE³⁴,(SEQ ID NO: 10) ¹⁷DPAVDLLKNYMQLGKQQREK³⁵, (SEQ ID NO: 11)¹⁷DPAVDLLKNYMQLGKQQREKQ³⁶, (SEQ ID NO: 12) ¹⁷DPAVDLLKNYMQLGKQQREKQ³⁷,(SEQ ID NO: 13) ¹⁷DPAVDLLKNYMQLGKQQREKQR³⁸,   (SEQ ID NO: 14)¹⁷DPAVDLLKNYMQLGKQQREKQRE³⁹, (SEQ ID NO: 15)¹⁷DPAVDLLKNYMQLGKQQREKQRES⁴⁰, (SEQ ID NO: 16)¹⁷DPAVDLLKNYMQLGKQQREKQRESR⁴¹, (SEQ ID NO: 17)¹⁷DPAVDLLKNYMQLGKQQREKQRESRE⁴², (SEQ ID NO: 18)¹⁷DPAVDLLKNYMQLGKQQREKQRESREK⁴³, (SEQ ID NO: 19)¹⁷DPAVDLLKNYMQLGKQQREKQRESREKP⁴⁴, (SEQ ID NO: 20)¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPY⁴⁵, (SEQ ID NO: 21)¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYK⁴⁶, (SEQ ID NO: 22)¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYKE⁴⁷, or (SEQ ID NO: 23)¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYKEV⁴⁸.

In a still further aspect, the minimal Vpx packaging motif consists of¹⁷DPAVDLLKNY²⁶ (SEQ ID NO: 1).

Chimeric vectors described herein may comprise SIVmac₂₃₉ nucleic acidsequences encoding the minimal Vpx packaging motif, wherein SIVmac₂₃₉nucleic acid sequences comprise at least one codon optimized nucleicacid sequence.

In a particular embodiment, the SIVmac₂₃₉ nucleic acid sequencesencoding the minimal Vpx packaging motif are inserted into the HIV-1nucleic acid sequences encoding p6 of HIV-1 Gag polyprotein.

In a more particular embodiment, the SIVmac₂₃₉ nucleic acid sequencesencoding the minimal Vpx packaging motif are inserted into the HIV-1nucleic acid sequences encoding p6 of HIV-1 Gag polyprotein to generatea hybrid HIV-1/SIVmac₂₃₉ nucleic acid sequence that encodes a hybridHIV-1/SIVmac₂₃₉ p6 wherein amino acids 1-14 of HIV-1 p6 are linkeddirectly to the minimal Vpx packaging motif consisting of at least 10contiguous amino acids of SIVmac₂₃₉ p6 comprising of ¹⁷DPAVDLLKNY²⁶ (SEQID NO: 1), wherein the 5′ terminus of the minimal Vpx packaging motif isthe aspartic acid (D) at amino acid position 17 of the SIVmac₂₃₉ p6amino acid sequence. See, for example, FIG. 1.

In another aspect, the chimeric vector comprises HIV-1 nucleic acidsequences that encode Gag and Pol.

In yet another aspect, the chimeric vector comprising HIV-1 nucleic acidsequences, wherein the HIV-1 nucleic acid sequences comprise HIV-1 p6 orpNL.Ba.L.

In a further aspect, a method of making a plurality of virions/viruseshaving enhanced infectivity for monocyte-derived macrophages (MDM) anddendritic cells (MDDC) is presented, the method comprising transfectinga population of cells with a lentiviral vector comprising 5′ and 3′ longterminal repeats (LTRs) and a nucleic acid sequence encoding at leastone immunogen of a peptide or protein, a vector encoding vesicularstomatitis virus (VSV) envelope glycoprotein, a vector encoding Vpx, anda chimeric vector comprising HIV-1 nucleic acid sequences and SIVmac₂₃₉nucleic acid sequences (as described herein), wherein the SIVmac₂₃₉nucleic acid sequences encode an SIVmac₂₃₉ amino acid sequenceconsisting of a minimal Vpx packaging motif that confers Vpx packagingactivity to the chimeric vector, to generate a transfected population ofcells, wherein the transfected population of cells produces theplurality of virions having enhanced infectivity for MDM and dendriticcells MDDC.

In an embodiment thereof, the vector comprising 5′ and 3′ LTRs furthercomprises a nucleic acid sequence encoding at least one dendritic cellactivator protein and/or at least one cytokine. An exemplary dendriticcell activator protein useful for such purposes is CD40 ligand (CD40L).Cytokines and cell surface proteins that would activate DC or could beexpressed in DCs to activate T cells include: IL-2 (GenBank No. S82692),IL-12 (GenBank No. NM_(—)000882), TNF-alpha (GenBank No. XO2910.1), andCD28 (GenBank No. NM_(—)006139.3).

In a particular aspect, the above method may further comprise the stepof administering the plurality of virions having enhanced infectivityfor MDM and dendritic cells MDDC or a composition thereof to a subjectin a therapeutically effective amount sufficient to enhance innateimmune responses to the peptide or protein in the subject. In aparticular embodiment thereof, the plurality of virions/virusescomprises at least one immunogen of an HIV-1 encoded peptide or protein,and the plurality of virions/viruses or the composition thereof isadministered to a subject in a therapeutically effective amountsufficient to enhance innate immune responses to HIV-1 in the subject.In a particular embodiment thereof, the subject is infected with HIV-1or suspected to be infected with HIV-1. In a more particular embodiment,the subject is a mammal. In an even more particular embodiment, themammal is a primate or a human.

Also encompassed herein is a plurality of virions/viruses havingenhanced infectivity for MDM and MDDC, which are suitable for use inhumans. As described herein, the plurality of virions/viruses havingenhanced infectivity for MDM and MDDC is produced by a method comprisingtransfecting a population of cells with a lentiviral vector comprising5′ and 3′ long terminal repeats (LTRs) and a nucleic acid sequenceencoding at least one immunogen of a peptide or protein, a vectorencoding vesicular stomatitis virus (VSV) envelope glycoprotein, avector encoding Vpx, and a chimeric vector comprising HIV-1 nucleic acidsequences and SIVmac₂₃₉ nucleic acid sequences (as described herein),wherein the SIVmac₂₃₉ nucleic acid sequences encode an SIVmac₂₃₉ aminoacid sequence consisting of a minimal Vpx packaging motif that confersVpx packaging activity to the chimeric vector, to generate a transfectedpopulation of cells, wherein the transfected population of cellsproduces the plurality of virions having enhanced infectivity for MDMand dendritic cells MDDC. Compositions comprising the plurality ofvirions/viruses having enhanced infectivity for MDM and MDDC andpharmaceutically acceptable carriers are also envisioned. The pluralityof virions/viruses made in accordance with the present method issuitable for use in humans at least in part because it is produced usingthe chimeric vector comprising HIV-1 nucleic acid sequences andSIVmac₂₃₉ nucleic acid sequences described herein.

In a further aspect, a method of enhancing innate immune responses to apeptide or protein in a subject is presented, the method comprising:administering the plurality of virions/viruses having enhancedinfectivity for MDM and MDDC or a composition thereof to the subject,wherein the plurality of virions/viruses comprises the at least oneimmunogen of the peptide or protein, and wherein the plurality ofvirions/viruses or a composition thereof is administered to the subjectin a therapeutically effective amount sufficient to enhance innateimmune responses to the peptide or protein in the subject. In aparticular embodiment, the subject is a mammal. In an even moreparticular embodiment, the mammal is a primate or a human.

In a still further aspect, a method of enhancing innate immune responsesto human immunodeficiency virus 1 (HIV-1) in a subject is presented, themethod comprising: administering the plurality of virions/viruses havingenhanced infectivity for MDM and MDDC or a composition thereof to thesubject, wherein the plurality of virions/viruses comprises the at leastone immunogen of the peptide or protein and the peptide or protein is anHIV-1 encoded peptide or protein, and wherein the plurality ofvirions/viruses or the composition thereof is administered to thesubject in a therapeutically effective amount sufficient to enhanceinnate immune responses to HIV-1 in the subject. In a particularembodiment thereof, the subject is infected with HIV-1 or suspected tobe infected with HIV-1. In a more particular embodiment, the subject isa mammal. In an even more particular embodiment, the mammal is a primateor a human.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. Identification of the minimal Vpx packaging motif in SIVmacp6 by transfer into HIV-1 p6. A. Alignment of NL4-3 and SIVmac₂₃₉ p6amino acid sequence. The two proposed alpha helices (α1 and α2) areshaded and the PTAPP late domain and TSG101 binding site are in boldunderline. Binding sites for ALIX and Vpx are in bold underline. Beloware diagrams of the HIV-1/SIVmac p6 chimeras with white for HIV-1 andblack for SIV sequence. The SIV sequence is inserted at position 14 ofHIV-1 p6, except in chimera 17-23(a), where the Vpx binding motif isdisplaced to position 21 to not alter the ¹⁴FRFG¹⁸ Vpr packaging motif.B. Immunoblot analysis shows packaging of SIVmac₂₃₉ Vpx in chimerascontaining the Vpx packaging motif 293T cells were cotransfected withpNL4-3 containing a wild-type or chimeric p6 and pcVpx.myc or emptyvector. Two days later, cell lysate and virions were pelleted byultracentrifugation and analyzed on an immunoblot. The immunoblot wasprobed with antibody to myc-tagged Vpx, HIV-1 p24 CA, or tubulin. ApcVpx-myc alone transfection was included to rule-out nonspecificrelease of Vpx. C. HIV-1 p6 chimeras that map amino acids required forVpx packaging. Virions were prepared by transfection and analyzed on animmunoblot as in (B). D. Effects of p6 mutations on packaging of HIV-1Vpr. 293T were cotransfected with wild-type or chimeric p6 pNL4-3 andpcVpr.myc or empty vector. Cell lysate and virions were analyzed on animmunoblot as in (B).

FIGS. 2A-C. Relative contribution of the two proposed Vpr packagingmotifs of HIV-1. A. Sequence of the two reported Vpr packaging motifs ofHIV-1 p6. The two motifs are underlined in bold. B. Immunoblot analysisof Vpr packaged by motif 1 mutant virions. Virions were generated bytransfection of 293T cells with pNL4-3 containing a wild-type or mutantp6 and a pcVpr.myc expression vector or empty vector. The virions werepelleted from the culture supernatants by ultracentrifugation andanalyzed on an immunoblot probed with antibody to myc-tagged Vpr, HIV-1CA p24, or tubulin. C. Immunoblot analysis of Vpr packaged by motif 2.Mutant virions prepared as in (B). M1A is F15, R16, F17, and G18 inmotif 1 mutated to alanine. M2Aa is L35, L38, L41, and L44 in motif 2mutated to alanine. M2Ab is M2Aa with the addition of S40, S43, and F45mutated to alanine. M1 and 2a is the combination of M1A and M2Aa. M1 and2b is the combination of M1A and M2Ab.

FIGS. 3A-C. Chimeric HIV-1 containing the Vpx packaging motif has anenhanced ability to infect MDDC and MDM. A. Expression and packaging ofVpx provided in trans. Luciferase reporter viruses were generated bycotransfection of 293T cells with wild-type or the 17-26 chimericproviral reporter virus plasmid (18 μs) and increasing amounts ofpcVpx.myc (0.3 μg, 0.6 μg, 3.0 μg, 6.0 μs) with the total mass of DNAheld constant by the addition of pcDNA plasmid. The resulting virionswere analyzed on an immunoblot probed with antibody to myc-tagged Vpx,HIV-1 CA p24, or tubulin. B. The effect of Vpx on MDDC and MDMinfection. MDDC (upper panel) and MDM (lower panel) were infected withVSV-G pseudotyped luciferase reporter viruses normalized for infectivityon 293T cells. After 4 days, the cultures were harvested and theluciferase activity was determined. The data are displayed as thefold-enhancement of the virus containing

Vpx divided by virus lacking Vpx. Error bars indicate the standarddeviation of triplicate measurements. Results from three MDDC and twoMDM donors are shown. C. P24 analysis of MDDC and MDM infection by HIV-1containing Vpx. MDDC (upper panels) and MDM (lower panels) were infectedwith luciferase reporter virus. Three days later, the cells werecollected and intracellular p24-FITC was determined by flow cytometry.

FIGS. 4A-B. Comparison of SIVmac Vpx, SIVagm Vpr, and HIV-2_(rod) Vpx.Luciferase reporter viruses were generated by cotransfection of 293Tcells with wild-type or p6 chimeric HIV-1 and expression vectors forSIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx, or SIVagm Vpr. A. Expression andpackaging of codon-optimized SIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx, and SIVagmVpr. Cell lysates and pelleted virions were analyzed on an immunoblotprobed with antibody to myc-tagged Vpx , myc-tagged Vpr, HIV-1 p24 CA,or tubulin. B. Effect of HIV-1 packaging SIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx,or SIVagm Vpr on MDDC and MDM infection. MDDC and MDM were infected withthe indicated virus, normalized by luciferase activity on 293T, and, 4days postinfection, luciferase activity was determined. Error barsindicate the standard deviation of triplicates. Three MDDC and MDMdonors are shown.

FIGS. 5A-C. CCR5-using chimeric virus with Vpx in cis replicates moreefficiently in MDDC and MDM. Wild-type and chimeric p6 NL.Ba.L virusesthat express Vpx in cis were generated by transfection of 293T cells. A.Immunoblot of SIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx and SIVagm Vpr expressed incis in the cell lysates of cells treated for 5 h with 20 μM MG132. Thecell lysates were analyzed on an immunoblot probed for myc-tagged Vpx,myc-tagged Vpr, or tubulin. B. The effect of p6 chimeric virus with vpxin cis on MDM infection. MDM were infected with wild-type or p6 chimericvpx in cis virus. After 24 and 48 h, total DNA was prepared and thereverse transcripts were quantified by qRT-PCR using primers specificfor the late products and 2-LTR circles. Early products were notquantified as these were found to be present in the virions prior toinfection as a result of endogenous reverse transcription. AZT (25 μM)was added to the sample infected with the p6 chimeric virus encoding Vpxand served to control for plasmid contamination and intra-virion reversetranscripts. C. MDDC infection by p6 chimeric virus with vpx or vpr incis. MDDC were infected with virus normalized for p24. After 3 days, thecells were collected, stained for intracellular p24 with anti-p24-FITC,and analyzed by flow cytometry. AZT (25 μM) was used to control forcontamination with input virus. Nelfinavir (3 μM) was used to limitreplication to a single cycle. Results from two donors are shown.

FIG. 6. Infection of MDDC with Vpx-containing virus stimulates an innateimmune response. MDDC from two healthy donors were infected with the p6chimeric virus complemented in trans with Vpx. AZT (25 μM) orRaltegravir (10 μM) was added to the indicated samples. The cultureswere harvested 24 and 48 h postinfection and IFN-β mRNA was quantifiedby qRT-PCR. The data are presented relative to G6PDH mRNA amplified inparallel.

FIGS. 7A-C. Exposure of MDDC to Vpx-containing virus allows forefficient transfer of virus to T cells. A. MDDC were infected withNL.Ba.1 wild-type or p6 chimeric virus containing vpx in cis and with orwithout additional Vpx complementation in trans. After 6 h, free viruswas removed, and after 48 h, CD3/CD28-activated autologous CD4 T cellswere added. Supernatant p24 was measured over 10 days. Results arerepresentative of MDDC and CD4 T cells from two donors. B. MDDC wereinfected as in (A). After 6 h, free virus was removed. After 48 h, mediaor CD3/CD28-activated autologous T cells were added. In parallel, Tcells alone were infected as in (A) and after 6 h, free virus wasremoved. Supernatant p24 was measured over 14 days. The results shownare representative of MDDC and CD4+ T cells from two donors. C. MDDC andT cells were infected as in (A) and cells were collected at day 3 postcoculture. The cells were incubated with anti-CD11c-APC andanti-p24-FITC and analyzed by flow cytometry. The cell populations weregated on CD11c and then evaluated for intracellular p24 using FACS.Result shown is representative of three donors tested.

FIG. 8. Nucleotide sequences of codon-optimized SIVmac239 Vpx, HIV-2rodVpx, and SIVagm Vpr open reading frames. Total length is indicatedfollowing the final base.

FIG. 9. P6 chimeric mutants package reduced amounts of HIV-1 Vpr. 293Twere cotransfected with wild-type or chimeric p6 pNL4-3 and pcVpr.myc orempty vector. Cell lysate and virions were analyzed on an immunoblotprobed with antibody against myc-tagged Vpr, HIV-1 p24 CA, or tubulin.

FIGS. 10A-C. Alteration of gag-pol to contain SIVp6 17-26 allows forpackaging of Vpx and enhanced infection of MDDC. pGK-NGFR-IRES GFPlentivirus was generated by cotransfecting 293T with the proviralplasmid pGK-NGFR-IRES-GFP, the wild-type or p6 chimeric gag/polpackaging plasmid (pMDLg/pRRE or pMDLg-SIVp6_(—)17-26, respectively),pcRSV-REV, pcVSV-G, and pcDNA6 or pcVpx.mychis. A. Immunoblot analysisof Vpx packaging by lentivirus with wild-type or p6 chimeric gag. Celllysates and pelleted virions were analyzed on an immunoblot probed withantibody to myc-tagged Vpx, HIV-2 p24 CA, or tubulin. B. Effect of Vpxpackaging on MDDC infection by pGK-NGFR-IRES-GFP. MDDC (1.25×10⁵) wereinfected with pGK-NGFR-IRES-GFP at 5 or 25 ng p24, and MDDC (3.0×10⁵)were infected with 120 ng p24. Three days after infection, cells werecollected and examined for GFP expression by FACS. C. Effect of Vpxpacking on 293T infection by pGK-NGFR-IRES-GFP. 293T (2.5×10⁵) wereinfected with pGK-NGFR-IRES-GFP at 120 ng p24. Three days afterinfection, cells were collected and examined for GFP expression by FACS.

FIG. 11 shows nucleic and amino acid sequences of NL4.3 Sequence—FullLength with Chimeric p6, 17-26

FIG. 12 shows nucleic and amino acid sequences of NL4.3 Sequence—FullLength, Wild Type

FIG. 13 shows SIVmac239 Vpx wild-type and codon-optimized sequences.

FIG. 14. The structure of LV vectors used to transduce DCs. The vectorsexpress a CMV promoter-driven CD40L, GFP or CD40-P2A-Flu fusion protein(CD40L-Flu) and a PGK-driven puromycin resistance gene or ER-retentionsignal linked to Flu matrix amino acids 58-66, GILGFVFTL (Chang et al.Methods in Molecular Biology 614:161-171, 2010), (Flu). The vectors wereproduced as viruses that contained or lacked Vpx by transfection of 293Tcells with the LV vector plasmid, the Gag/Pol packaging vector, HIV-1Rev, and SIVmac Vpx expression vector pcVpx and pseudotyped with VSV-G.

FIG. 15. Packaged Vpx allows for efficient DC transduction. DCs weretransduced with LV vector indicated below each panel. Transductionefficiency was determined by analysis of GFP and CD40L expression. (Vpx+constructs on top).

FIGS. 16A-B. Transduction of DCs with Vpx-containing vectors expressingCD40L induces DC maturation. DCs were transduced with the LV vectorsthat contained or lacked Vpx. After 48 and 72 h, the number of cellsthat expressed CD83 and CD86 were determined by flow cytometry. A. CD83expression at 72 h post-transduction (Vpx+ constructs on top). CD83expression for each infected DC population (blue) is contrasted againstuninfected DCs (red). B. CD83 expression on the DC of four donors

FIGS. 17A-B. Transduction of DCs with Vpx-containing LV vectorsexpressing CD40L induces a high level of IL-12. Cytokine bead arrayanalysis on culture supernatants shows the IL-12 levels secreted by DCsfrom four different donors at 72 h post transduction with different LVconstructs as compared to IL-12 levels secreted from uninfected DCs.

FIG. 18. DCs transduced with peptide and CD40L expressing lentiviralvectors activate a CTL response. Transduced DCs were cultured with CTLclone. After 24 h, the supernatant IFN-γ was quantified by CBA. As acontrol, the cells were pulsed with synthetic flu peptide, T cellsalone, T cell clones co-cultured with uninfected DCs, and T cell clonesco-cultured with uninfected DCs and then pulsed with synthetic flupeptide.

FIG. 19. DCs transduced with Vpx-containing LV vectors encoding CD40Land a peptide epitope from influenza enhance antigen-specific CD8+memory T cell responses. To assess for a Flu-specific memory response,flu-specific tetramer staining was performed on autologous CD8+CD3+ Tcells two weeks after being co-cultured with DCs transduced by differentLV vector constructs (Vpx+ constructs on top). Analysis on T cellsco-cultured with uninfected DC and uninfected DC pulsed with syntheticflu peptide were performed as controls.

FIG. 20. DCs transduced with CD40L expressing lentiviral vectorspackaged with Vpx induce reactivation of latent HIV from ACH-2 cells.DCs were transduced with indicated LV vector and then co-cultured withACH-2 cells. Infectious virus in the supernatant was quantified byTZM-bl assay to assess reactivation of HIV-1 proviruses.

DETAILED DESCRIPTION OF THE INVENTION

The lentiviral Vpx accessory protein is thought to facilitate theinfection of macrophages and dendritic cells by counteracting a yetunidentified host restriction. Although HIV-1 does not encode Vpx, itcan be provided to monocyte-derived macrophages (MDM) andmonocyte-derived dendritic cells (MDDC) through virus-like particles todramatically enhance their susceptibility to HIV-1. Vpx and the relatedaccessory protein Vpr are packaged into virions through a virus-specificinteraction with the p6 carboxy-terminal domain of Gag. Here, thepresent inventors determined the minimal Vpx packaging motif ofSIVmac₂₃₉ p6 and introduced this ten amino acid sequence into the p6region of an infectious HIV-1 molecular clone. The chimeric virusefficiently packaged Vpx provided in trans and was substantially moreinfectious on MDDC and MDM. The present inventors further engineered thevirus to express Vpx in cis by introducing the coding sequence in placeof nef. The resulting virus produced less Vpx but was significantlyenhanced in its infectivity on MDDC and MDM. Infection of the cells withVpx-containing HIV-1 induced a potent type I interferon response. In aco-culture system, Vpx-containing HIV-1 was enhanced in its ability tobe transmitted from MDDC to T cells. These findings suggest that Vpxcould facilitate dendritic cell to T cell virus transmission in vivo.See, for example, Examples 1-2 herein below. The chimeric virusesdescribed herein are envisioned as useful for the design of dendriticcell vaccines that induce an innate immune response in MDDC.Additionally, this approach can be useful for designing and generatingengineered lentiviral vectors to produce virions that can be used totransduce these relatively resistant cells.

Further to the above, dendritic cells (DCs) are central to the inductionof innate and adaptive immune responses yet are difficult to manipulatefor therapeutic purposes due to their resistance to the introduction oftransfection or transduction. Lentiviral vectors offer the ability toexpress genes stably in cells but are restricted in DCs by SAMHD1, ahost factor that blocks retroviruses at reverse transcription. Examples1 and 2 illustrate that the present inventors have developed lentiviralvectors and have used same to produce lentiviral vector virions thatescape the restriction in DCs by virtue of packaging the SIV accessoryprotein Vpx. Such lentiviral vector virions comprise the lentiviralvector used to produce the virions. As a proof of principal, the presentinventors have constructed lentiviral vectors that express an HLAA2-restricted influenza (Flu) peptide and immunostimulatory CD40L. Asdescribed in Example 3, the Flu peptide was expressed as a fusionprotein with CD40L separated by the P2A self-cleaving peptide. BecauseCD40L is a type II transmembrane protein, this configuration places thecarboxy-terminal Flu peptide in the endoplasmic reticulum (ER) where itcan be shunted into the antigen presentation pathway duringbiosynthesis. The Vpx-containing vectors infected DCs 60-fold betterthan control Vpx-negative viruses. Vpx-containing vectors induced theDCs to mature as judged by CD83 and CD86 upregulation and inducedTh1-skewing inflammatory cytokines. The transduced DCs, moreover,stimulated a Flu-peptide-specific CD8+ CTL clone and antigen-specificCD8+ cells in PBMC from healthy human donors, as assessed by IFNγ andtetramer staining. This system, therefore, provides the basis for thedevelopment of an effective DC-targeted anti-HIV-1 vaccine.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A. Terminology

The term “specific binding member” describes a member of a pair ofmolecules which have binding specificity for one another. The members ofa specific binding pair may be naturally derived or wholly or partiallysynthetically produced. One member of the pair of molecules has an areaon its surface, or a cavity, which specifically binds to and istherefore complementary to a particular spatial and polar organisationof the other member of the pair of molecules. Thus the members of thepair have the property of binding specifically to each other. Examplesof types of specific binding pairs are antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate. Thisapplication is concerned with antigen-antibody type reactions.

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antibody binding domain. CDR grafted antibodies arealso contemplated by this term. An “antibody” is any immunoglobulin,including antibodies and fragments thereof, that binds a specificepitope. The term encompasses polyclonal, monoclonal, and chimericantibodies, the last mentioned described in further detail in U.S. Pat.Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wildtype immunoglobulin (Ig) molecule, generally comprising four full lengthpolypeptide chains, two heavy (H) chains and two light (L) chains, or anequivalent Ig homologue thereof (e.g., a camelid nanobody, whichcomprises only a heavy chain); including full length functional mutants,variants, or derivatives thereof, which retain the essential epitopebinding features of an Ig molecule, and including dual specific,bispecific, multispecific, and dual variable domain antibodies;Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD,IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, andIgA2). Also included within the meaning of the term “antibody” is any“antibody fragment”.

An “antibody fragment” means a molecule comprising at least onepolypeptide chain that is not full length, including (i) a Fab fragment,which is a monovalent fragment consisting of the variable light (VL),variable heavy (VH), constant light (CL) and constant heavy 1 (CH1)domains; (ii) a F(ab′)2 fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a heavy chain portion of an Fab (Fd) fragment, whichconsists of the VH and CH1 domains; (iv) a variable fragment (Fv)fragment, which consists of the VL and VH domains of a single arm of anantibody, (v) a domain antibody (dAb) fragment, which comprises a singlevariable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) acamelid antibody; (vii) an isolated complementarity determining region(CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VLdomain are linked by a peptide linker which allows the two domains toassociate to form an antigen binding site (Bird et al, Science, 242,423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) adiabody, which is a bivalent, bispecific antibody in which VH and VLdomains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with the complementaritydomains of another chain and creating two antigen binding sites(WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448,(1993)); and (x) a linear antibody, which comprises a pair of tandem Fvsegments (VH-CH1-VH-CH1) which, together with complementarity lightchain polypeptides, form a pair of antigen binding regions; (xi)multivalent antibody fragments (scFv dimers, trimers and/or tetramers(Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); and (xii)other non-full length portions of heavy and/or light chains, or mutants,variants, or derivatives thereof, alone or in any combination.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of light chain or heavy and light chain variable andhypervariable regions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Antibodies may also be bispecific, wherein one binding domain of theantibody is a specific binding member of the invention, and the otherbinding domain has a different specificity, e.g. to recruit an effectorfunction or the like. Bispecific antibodies of the present inventioninclude wherein one binding domain of the antibody is a specific bindingmember of the present invention, including a fragment thereof, and theother binding domain is a distinct antibody or fragment thereof,including that of a distinct anti-cancer or anti-tumor specificantibody. The other binding domain may be an antibody that recognizes ortargets a particular cell type, as in a neural or glial cell-specificantibody. In the bispecific antibodies of the present invention the onebinding domain of the antibody of the invention may be combined withother binding domains or molecules which recognize particular cellreceptors and/or modulate cells in a particular fashion, as for instancean immune modulator (e.g., interleukin(s)), a growth modulator orcytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNFbispecific modality demonstrated in U.S. Ser. No. 60/355,838 filed Feb.13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin)or anti-mitotic or apoptotic agent or factor.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may alsocontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “antigen binding domain” describes the part of an antibodywhich comprises the area which specifically binds to and iscomplementary to part or all of an antigen. Where an antigen is large,an antibody may bind to a particular part of the antigen only, whichpart is termed an epitope. An antigen binding domain may be provided byone or more antibody variable domains. Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show any significant bindingto molecules other than its specific binding partner(s). The term isalso applicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response, particularly to an antigen. An adjuvant can serve as atissue depot that slowly releases the antigen and also as a lymphoidsystem activator that non-specifically enhances the immune response(Hood et al., Immunology, Second Ed, 1984, Benjamin/Cummings: MenloPark, Calif., p. 384). Often, a primary challenge with an antigen alone,in the absence of an adjuvant, will fail to elicit a humoral or cellularimmune response. Previously known and utilized adjuvants include, butare not limited to, complete Freund's adjuvant, incomplete Freund'sadjuvant, saponin, mineral gels such as aluminum hydroxide, surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvant such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Mineral saltadjuvants include but are not limited to: aluminum hydroxide, aluminumphosphate, calcium phosphate, zinc hydroxide and calcium hydroxide.Preferably, the adjuvant composition further comprises a lipid of fatemulsion comprising about 10% (by weight) vegetable oil and about 1-2%(by weight) phospholipids. Preferably, the adjuvant composition furtheroptionally comprises an emulsion form having oily particles dispersed ina continuous aqueous phase, having an emulsion forming polyol in anamount of from about 0.2% (by weight) to about 49% (by weight),optionally a metabolizable oil in an emulsion-forming amount of up to15% (by weight), and optionally a glycol ether-based surfactant in anemulsion-stabilizing amount of up to about 5% (by weight).

As used herein, the term “immunomodulator” refers to an agent which isable to modulate an immune response. An example of such modulation is anenhancement of cell activation or of antibody production.

The term “effective amount” of an immunomodulator refers to an amount ofan immunomodulator sufficient to enhance a vaccine-induced immuneresponse, be it cell-mediated, humoral or antibody-mediated. Aneffective amount of an immunomodulator, if injected, can be in the rangeof about 0.1-1,000 μg, preferably 1-900 μg, more preferably 5-500 μg,for a human subject, or in the range of about 0.01-10.0 μg/Kg bodyweight of the subject animal. This amount may vary to some degreedepending on the mode of administration, but will be in the same generalrange. If more than one immunomodulator is used, each one may be presentin these amounts or the total amount may fall within this range. Aneffective amount of an antigen may be an amount capable of eliciting ademonstrable immune response in the absence of an immunomodulator. Formany antigens, this is in the range of about 5-100 μg for a humansubject. The appropriate amount of antigen to be used is dependent onthe specific antigen and is well known in the art.

The exact effective amount necessary will vary from subject to subject,depending on the species, age and general condition of the subject, theseverity of the condition being treated, the mode of administration,etc. Thus, it is not possible to specify an exact effective amount.However, the appropriate effective amount may be determined by one ofordinary skill in the art using only routine experimentation or priorknowledge in the vaccine art.

An “immunological response” to a composition or vaccine comprised of anantigen is the development in the host of a cellular- and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, such a response consists of the subject producingantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

The term “immunogen” refers to a substance that provokes an immuneresponse.

The term “comprise” is generally used in the sense of include, that isto say permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention referred to above, those of skill in the art willappreciate that minor modifications to the N- or C-terminal of thepeptide may however be contemplated, such as the chemical modificationof the terminal to add a protecting group or the like, e.g. theamidation of the C-terminus.

The term “isolated” refers to the state in which specific bindingmembers of the invention, or nucleic acid encoding such binding memberswill be, in accordance with the present invention. Members and nucleicacid will be free or substantially free of material with which they arenaturally associated such as other polypeptides or nucleic acids withwhich they are found in their natural environment, or the environment inwhich they are prepared (e.g. cell culture) when such preparation is byrecombinant DNA technology practised in vitro or in vivo. Members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtitre platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfuctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding protein or peptide sequences asprovided herein, or comprising sequences which are degenerate thereto.DNA sequences having the nucleic acid sequence encoding the peptides ofthe invention are contemplated, including degenerate sequences thereofencoding the same, or a conserved or substantially similar, amino acidsequence. By “degenerate to” is meant that a different three-lettercodon is used to specify a particular amino acid. It is well known inthe art that the following codons can be used interchangeably to codefor each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in the sequences encoding the protein or peptidesequences of the invention, such that a particular codon is changed to acodon which codes for a different amino acid. Such a mutation isgenerally made by making the fewest nucleotide changes possible. Asubstitution mutation of this sort can be made to change an amino acidin the resulting protein in a non-conservative manner (i.e., by changingthe codon from an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic to an amino acid belonging toanother grouping) or in a conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to the samegrouping). Such a conservative change generally leads to less change inthe structure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein. Further, variants and subtypes of HIV are known andrecognized and any such variants or subtype corresponding protein orpeptide sequences of the invention (e.g., Vpx) are encompassed andcontemplated herein.

Codon-optimized forms of vpx, for example, are used to advantage in thechimeric viruses and lentiviral vectors described herein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino acids with uncharged polar R groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino acids with charged polar R groups (negatively charged at Ph 6.0)Aspartic acid, Glutamic acidBasic amino acids (positively charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free -OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Exemplary and preferred conservative amino acid substitutions includeany of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L)for valine (V) and vice versa; serine (S) for threonine (T) and viceversa; isoleucine (I) for valine (V) and vice versa; lysine (K) forglutamine (Q) and vice versa; isoleucine (I) for methionine (M) and viceversa; serine (S) for asparagine (N) and vice versa; leucine (L) formethionine (M) and vice versa; lysine (L) for glutamic acid (E) and viceversa; alanine (A) for serine (S) and vice versa; tyrosine (Y) forphenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid(D) and vice versa; leucine (L) for isoleucine (I) and vice versa;lysine (K) for arginine (R) and vice versa.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds or drugcandidate compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense or stimulates a response thatwould be elicited on binding of a natural binder to a binding site.

The term ‘assay’ means any process used to measure a specific propertyof a compound or agent. A ‘screening assay’ means a process used tocharacterize or select compounds based upon their activity from acollection of compounds.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

‘Therapeutically effective amount’ means that amount of a drug,compound, antimicrobial, antibody, or pharmaceutical agent that willelicit the biological or medical response of a subject that is beingsought by a medical doctor or other clinician. As an example, withregard to immune response, the term “effective amount” is intended toinclude an effective amount of a compound or agent that will bring abouta biologically meaningful increase in the amount of or extent of immuneresponse, activation indicator and/or a biologically meaningful increasein the amount or extent of dendritic cell, T cell and/or B cell effects.The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to promote, and preferably increase by at least about30 percent, more preferably by at least 50 percent, most preferably byat least 90 percent, a clinically significant change in the immuneresponse or immune cell indicator or response, or in a patient'sresponse to an antigen, vaccine, or other immune agent, or in apatient's clearance of an infectious agent, or other feature ofpathology such as for example, elevated activated T or B cells,activated DC cell count, fever or white cell count.

The term ‘treating’ or ‘treatment’ of any disease or infection refers,in one embodiment, to ameliorating the disease or infection (i.e.,arresting the disease or growth of the infectious agent or bacteria orreducing the manifestation, extent or severity of at least one of theclinical symptoms thereof). In another embodiment ‘treating’ or‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or infection,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, ‘treating’ or ‘treatment’ relates to slowingthe progression of a disease or reducing an infection.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

As used herein, the term “replication defective HIV vector” refers to avector that encodes HIV peptides/proteins, but does not encode livevirus.

HIV-1 comprises several major genes that encode structural proteins thatare common to all lentiviruses and several nonstructural genes. The gag(group specific antigen) gene encodes the Gag polyprotein, which isprocessed during maturation to matrix protein (MA, p17), capsid protein(CA, p24), spacer peptide 1 (SP1, p2), nucleocapsid protein (NC, p7),spacer peptide 2 (SP2, p 1) and p6. These proteins provide for thephysical infrastructure of the virus. The pol gene encodes viral enzymesfor reverse transcriptase (RT), integrase (IN), and HIV protease (PR).

B. Detailed Disclosure

Lentiviral vectors are currently used to stably express proteins andsmall RNAs in a variety of cell types, including primary cells. However,myeloid cells, such as macrophages and dendritic cells, are relativelyresistant to viral transduction by current lentiviral systems.Macrophages and dendritic cells are initiators of adaptive immuneresponses that confer immunity during vaccination and also mediateinnate immune responses to pathogens. A lentiviral vector thatefficiently transduces this cell type would facilitate improvedexpression of therapeutic proteins and RNAs in macrophages and dendriticcells. Methods using such lentiviral vectors would, moreover, be usefulin the design of vaccines against HIV and/or other pathogens to induce astronger immune response to the immunogen.

Vpx is a lentiviral accessory protein encoded by a subset oflentiviruses, including SIVmac of the rhesus macaque, but is absent fromHIV-1. Consequently, the Vpx protein is packaged in SIV virions, but notin those of HIV-1 or lentiviral vectors based on HIV-1. In SIVmac, Vpxgreatly enhances the ability of the virus to infect MDM and MDDC.

The invention described herein allows Vpx to be delivered and packagedinto HIV-1 and HIV-1 derived lentiviral vector virions. To allow for Vpxpackaging, the present inventors altered the p6 region of HIV-1 toencode a ten amino acid sequence in p6 of SIVmac₂₃₉. As defined herein,the ten amino acid sequence (¹⁷DPAVDLLKNY²⁶) is the minimal sequencerequired for Vpx packaging. To generate the modified virions, 293 cellswere transfected with lentiviral vector DNA, VSV-G expression plasmidand p6-chimeric HIV-1 Gag/pol expression vector. After 3 days,virus-containing culture supernatant is collected and frozen for lateruse. Details pertaining to making these constructs and virions and theircomposition are presented in, for example, the Drawings and Examples.

The constructs, vectors, and virions described herein can be used toimprove the efficiency of transduction of myeloid cells by lentiviralvectors. The virions contain high copy numbers of functional Vpx,increasing their infectivity 100-fold in MDM and MDDC.

The present constructs, vectors, and virions differ in many respectswhen compared to those generated by other investigators. Theintroduction of the minimal Vpx packaging motif into HIV-1 basedlentiviral vectors generates a new genus of lentiviral vector havingunique structural features (e.g., nucleic acid sequences) that confernew and surprising functional properties, predominant among theseproperties is the enhanced ability of HIV-1 virions generated therefromto infect MDM and MDDC. More particularly, the presence of the minimalVpx packaging motif in the new genus of HIV-1 based lentiviral vectorsmakes it possible to package Vpx supplied in trans into virus likeparticles generated therefrom that can promote HIV-1 infection of MDMand MDDC. In a further embodiment, the present inventors have modifiedthe new genus of HIV-1 based lentiviral vectors comprising the minimalVpx packaging motif to include SIV vpx. Accordingly, in a secondgeneration of HIV-1 based lentiviral vectors, Vpx is supplied in cis,thus eliminating the need for additional constructs/vectors/VLPsencoding Vpx. Methods described herein thus produce lentiviral vectorparticles which comprise packaged Vpx protein.

The methods described herein can be used to generate lentiviral vectorsthat express a protein or RNA of interest that have an improved abilityto infect MDM and MDDC. This is useful for basic research into signaltransduction pathways in these specific cells types. The approach mayalso be used advantageously in the clinic for gene therapy to achievestable expression of protein or RNA in vivo for the treatment of geneticdeficiencies.

The method may also find application for vaccination approaches thatrely on transduction of MDM and MDDC. The chimeric vectors, constructs,and VLPs described herein will enhance the delivery and subsequentexpression of antigens in such cells and thus, lead to more effectivevaccine responses. Indeed, current efforts to generate dendriticcell-based cancer vaccines would be significantly enhanced byincorporating the chimeric vectors and constructs described herein.

Both HIV-1 p6 and pNL.Ba.L are identical to the HIV-1 reference strainNL4-3, as present in Genbank and presented herein. The essential genesof HIV-1 p6 and pNL.Ba.L with respect to the present chimeric vectorsand constructs include the following:

GAG Polyprotein Precursor: 168-1670 Nucleotide Sequence (SEQ ID NO: 24)atg ggtgcgagag cgtcggtatt aagcggggga gaattagata aatgggaaaa aattcggtta aggccagggggaaagaaaca atataaacta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggccttttagagacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcattatataataca atagcagtcc tctattgtgt gcatcaaagg atagatgtaa aagacaccaa ggaagcctta gataagatagaggaagagca aaacaaaagt aagaaaaagg cacagcaagc agcagctgac acaggaaaca acagccaggt cagccaaaattaccctatag tgcagaacct ccaggggcaa atggtacatc aggccatatc acctagaact ttaaatgcat gggtaaaagtagtagaagag aaggctttca gcccagaagt aatacccatg ttttcagcat tatcagaagg agccacccca caagatttaaataccatgct aaacacagtg gggggacatc aagcagccat gcaaatgtta aaagagacca tcaatgagga agctgcagaatgggatagat tgcatccagt gcatgcaggg cctattgcac caggccagat gagagaacca aggggaagtg acatagcaggaactactagt acccttcagg aacaaatagg atggatgaca cataatccac ctatcccagt aggagaaatc tataaaagatggataatcct gggattaaat aaaatagtaa gaatgtatag ccctaccagc attctggaca taagacaagg accaaaggaaccctttagag actatgtaga ccgattctat aaaactctaa gagccgagca agcttcacaa gaggtaaaaa attggatgacagaaaccttg ttggtccaaa atgcgaaccc agattgtaag actattttaa aagcactggg accaggagcg acactagaagaaatgatgac agcatgtcag ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag ccaagtaacaaatccagcta ccataatgat acagaaaggc aattttagga accaaagaaa gactgttaag tgtttcaatt gtggcaaagaagggcacata gccaaaaatt gcagggcccc taggaaaaag ggctgttgga aatgtggaaa ggaaggacac caaatgaaagattgtactga gagacaggct aattttttag ggaagatctg gccttcccac aagggaaggc cagggaattt tcttcagagcagaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga gacaacaact ccctctcaga ggcaggagccgatagacaag gaactgtatc ctttagcttc cctcagatca ctctttggca gcgacccctc gtcacaataa agatagggggTranslation (SEQ ID NO: 25)MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKIEEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQRQEPIDKELYPLASLRSLFGSDPSSQPOL Polyprotein Precursor: 1463-4474 Nucleotide Sequence (SEQ ID NO: 26)ttttttag ggaagatctg gccttcccac aagggaaggc cagggaattt tcttcagagc agaccagagc caacagccccaccagaagag agcttcaggt ttggggaaga gacaacaact ccctctcaga ggcaggagcc gatagacaag gaactgtatcctttagcttc cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg gcaattaaag gaagctctattagatacagg agcagatgat acagtattag aagaaatgaa tttgccagga agatggaaac caaaaatgat agggggaattggaggtttta tcaaagtaag acagtatgat cagatactca tagaaatctg cggacataaa gctataggta cagtattagtaggacctaca cctgtcaaca taattggaag aaatctgttg actcagattg gctgcacttt aaattttccc attagtcctattgagactgt accagtaaaa ttaaagccag gaatggatgg cccaaaagtt aaacaatggc cattgacaga agaaaaaataaaagcattag tagaaatttg tacagaaatg gaaaaggaag gaaaaatttc aaaaattggg cctgaaaatc catacaatactccagtattt gccataaaga aaaaagacag tactaaatgg agaaaattag tagatttcag agaacttaat aagagaactcaagatttctg ggaagttcaa ttaggaatac cacatcctgc agggttaaaa cagaaaaaat cagtaacagt actggatgtgggcgatgcat atttttcagt tcccttagat aaagacttca ggaagtatac tgcatttacc atacctagta taaacaatgagacaccaggg attagatatc agtacaatgt gcttccacag ggatggaaag gatcaccagc aatattccag tgtagcatgacaaaaatctt agagcctttt agaaaacaaa atccagacgt agtcatctat caatacatgg atgatttgta tgtaggatctgacttagaaa tagggcagca tagaacaaaa atagaggaac tgagacaaca tctgttgagg tggggattta ccacaccagacaaaaaacat cagaaagaac ctccattcct ttggatgggt tatgaactcc atcctgataa atggacagta cagcctatagtgctgccaga aaaggacagc tggactgtca atgacataca gaaattagtg ggaaaattga attgggcaag tcagatttatgcagggatta aagtaaggca attatgtaaa cttcttaggg gaaccaaagc actaacagaa gtagtaccac taacagaagaagcagagcta gaactggcag aaaacaggga gattctaaaa gaaccggtac atggagtgta ttatgaccca tcaaaagacttaatagcaga aatacagaag caggggcaag gccaatggac atatcaaatt tatcaagagc catttagaaa tctgaaaacaggaaagtatg caagaatgaa gggtgcccac actaatgatg tgaaacaatt aacagaggca gtacaaaaaa tagccacagaaagcatagta atatggggaa agactcctaa atttaaatta cccatacaaa aggaaacatg ggaagcatgg tggacagagtattggcaagc cacctggatt cctgagtggg agtttgtcaa tacccctccc ttagtgaagt tatggtacca gttagagaaagaacccataa taggagcaga aactttctat gtagatgggg cagccaatag ggaaactaaa ttaggaaaag caggatatgtaactgacaga ggaagacaaa aagttgtccc cctaacggac acaacaaatc agaagactga gttacaagca attcatctagctttgcagga ttcgggatta gaagtaaaca tagtgacaga ctcacaatat gcattgggaa tcattcaagc acaaccagataagagtgaat cagagttagt cagtcaaata atagagcagt taataaaaaa ggaaaaagtc tacctggcat gggtaccagcacacaaagga attggaggaa atgaacaagt agataaattg gtcagtgctg gaatcaggaa agtactattt ttagatggaatagataaggc ccaagaagaa catgagaaat atcacagtaa ttggagagca atggctagtg attttaacct accacctgtagtagcaaaag aaatagtagc cagctgtgat aaatgtcagc taaaagggga agccatgcat ggacaagtag actgtagcccaggaatatgg cagctagatt gtacacattt agaaggaaaa gttatcttgg tggcagttca tgtagccagt ggatatatagaagcagaagt aattccagca gagacagggc aagaaacagc atacttcctc ttaaaattag caggaagatg gccagtaaaaacagtacata cagacaatgg cagcaatttc accagtacta cagttaaggc cgcctgttgg tgggcgggaa tcaagcaggaatttggcatt ccctacaatc cccaaagtca aggagtaata gaatctatga ataaagaatt aaagaaaatt ataggacaggtaagagatca ggctgaacat cttaagacag cagtacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggattggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattacaaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggaaaggacc agcaaagctc ctctggaaaggtgaaggggc agtagtaata caagataata gtgacataaa agtagtgcca agaagaaaag caaagatcat cagggattatggaaaacaga tggcaggtga tgattgtgtg gcaagtagac aggatgagga ttaa Translation(SEQ ID NO: 27)FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDVVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKINGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFRNLKTGKYARMKGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED VIF Protein: 4419-4997 Nucleotide Sequence(SEQ ID NO: 28)at ggaaaacaga tggcaggtga tgattgtgtg gcaagtagac aggatgagga ttaacacatg gaaaagattagtaaaacacc atatgtatat ttcaaggaaa gctaaggact ggttttatag acatcactat gaaagtacta atccaaaaataagttcagaa gtacacatcc cactagggga tgctaaatta gtaataacaa catattgggg tctgcataca ggagaaagagactggcattt gggtcaggga gtctccatag aatggaggaa aaagagatat agcacacaag tagaccctga cctagcagaccaactaattc atctgcacta ttttgattgt ttttcagaat ctgctataag aaataccata ttaggacgta tagttagtcctaggtgtgaa tatcaagcag gacataacaa ggtaggatct ctacagtact tggcactagc agcattaata aaaccaaaacagataaagcc acctttgcct agtgttagga aactgacaga ggacagatgg aacaagcccc agaagaccaa gggccacagagggagccata caacgaatgg acactag Translation (SEQ ID NO: 29)MENRWQVMIVWQVDRMRINTWKRLVKHHMYISRKAKDWFYRHHYESTNPKISSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKKRYSTQVDPDLADQLIHLHYFDCFSESAIRNTILGRIVSPRCEYQAGHNKVGSLQYLALAALIKPKQIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTTNGH VPR Protein: 4937-5227 Nucleotide Sequence(SEQ ID NO: 30)atgg aacaagcccc agaagaccaa gggccacaga gggagccata caacgaatgg acactagagc ttttagaggaacttaagagt gaagctgtta gacattttcc taggatatgg ctccataact taggacgaca tatctatgaa acttacggggatacttgggc aggagtggaa gccataataa gaattctgca acaactgccg tttatccatt tcagaattgg gtgtcgacatagcagaatag gcgttactcg acagaggaga gcaagaaatg gagccagtag atcctagTranslation (SEQ ID NO: 31)MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQSRGDPTGPKETAT Protein: 5208-7792 (translate 5208-5422, 7747-7792)Nucleotide Sequence (SEQ ID NO: 32)atg gagccagtag atcctagact agagccctgg aagcatccag gaagtcagcc taaaactgct tgtaccaattgctattgtaa aaagtgttgc tttcattgcc aagtttgttt catgacaaaa gccttaggca tctcctatgg caggaagaagcggagacagc gacgaagagc tcatcagaac agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaatgcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata gtaatcatagaatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaa gagcagaaga cagtggcaatgagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggata ttgatgatctgtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaag caaccaccac tctattttgtgcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgt gtacccacag accccaacccacaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggt agaacagatg catgaggatataatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgtta gtttaaagtg cactgatttgaagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagata aaaaactgct ctttcaatatcagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatat agtaccaata gataataccagctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat ccccatacattattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaaca ggaccatgta caaatgtcagcacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatgg cagtctagca gaagaagatgtagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaaca catctgtaga aattaattgtacaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagca tttgttacaa taggaaaaataggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaa acagatagct agcaaattaagagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccag aaattgtaac gcacagttttaattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggttt aatagtactt ggagtactgaagggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatt tataaacatg tggcaggaagtaggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatatta ctgggctgct attaacaagagatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagg gacaattgga gaagtgaattatataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaaaaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacgctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctg agggctattg aggcgcaacagcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatcaacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaataaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaac aattacacaa gcttaatacactccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgtggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaagaatagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaatcccgagggga cccgacaggc ccgaaggaat ag Translation (SEQ ID NO: 33)MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQSRGDPTGPKEREV Protein: 5347-8021 (translate 5347-5422, 7747-8021)Nucleotide Sequence (SEQ ID NO: 34)atgg caggaagaag cggagacagc gacgaagagc tcatcagaac agtcagactc atcaagcttc tctatcaaagcagtaagtag tacatgtaat gcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgtgtggtccata gtaatcatag aatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaagagcagaaga cagtggcaat gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgctccttgggata ttgatgatct gtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaagcaaccaccac tctattttgt gcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgtgtacccacag accccaaccc acaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggtagaacagatg catgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgttagtttaaagtg cactgatttg aagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagataaaaaactgct ctttcaatat cagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatatagtaccaata gataatacca gctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcctttgagccaat ccccatacat tattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaacaggaccatgta caaatgtcag cacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatggcagtctagca gaagaagatg tagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaacacatctgtaga aattaattgt acaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagcatttgttacaa taggaaaaat aggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaaacagatagct agcaaattaa gagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccagaaattgtaac gcacagtttt aattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggtttaatagtactt ggagtactga agggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatttataaacatg tggcaggaag taggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatattactgggctgct attaacaaga gatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagggacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagagaagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcactatgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctgagggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgtggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgccttggaatgctag ttggagtaat aaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaacaattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaattagataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtaggaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcgtttcagaccc acctcccaat cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gaggcagagacagatccatt cgattagtga acggatcctt agcacttatc tgggacgatc tgcggagcct gtgcctcttc agctaccaccgcttgagaga cttactcttg attgtaacga ggattgtgga acttctggga cgcagggggt gggaagccct caaatattggtggaatctcc tacagtattg gagtcaggaa ctaaagaata g Translation(SEQ ID NO: 35)MAGRSGDSDEELIRTVRLIKLLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTVLESGTKE VPU Protein: 5439-5684 Nucleotide Sequence(SEQ ID NO: 36)at gcaacctata atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccatagtaatcatag aatataggaa aatattaaga caaagaaaaa tagacaggtt aattgataga ctaatagaaa gagcagaagacagtggcaat gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggatattgatgatct gtag Translation (SEQ ID NO: 37)MQPIIVAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEVSALVEMGVEMGHHAPWDIDDLENV Polyprotein Precursor: 5599-8163 Nucleotide Sequence (SEQ ID NO: 38)at gagagtgaag gagaagtatc agcacttgtg gagatggggg tggaaatggg gcaccatgct ccttgggatattgatgatct gtagtgctac agaaaaattg tgggtcacag tctattatgg ggtacctgtg tggaaggaag caaccaccactctattttgt gcatcagatg ctaaagcata tgatacagag gtacataatg tttgggccac acatgcctgt gtacccacagaccccaaccc acaagaagta gtattggtaa atgtgacaga aaattttaac atgtggaaag atgacatggt agaacagatgcatgaggata taatcagttt atgggatcaa agcctaaagc catgtgtaaa attaacccca ctctgtgtta gtttaaagtgcactgatttg aagaatgata ctaataccaa tagtagtagc gggagaatga taatggagaa aggagagata aaaaactgctctttcaatat cagcacaagc ataagagata aggtgcagaa agaatatgca ttcttttata aacttgatat agtaccaatagataatacca gctataggtt gataagttgt aacacctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaatccccatacat tattgtgccc cggctggttt tgcgattcta aaatgtaata ataagacgtt caatggaaca ggaccatgtacaaatgtcag cacagtacaa tgtacacatg gaatcaggcc agtagtatca actcaactgc tgttaaatgg cagtctagcagaagaagatg tagtaattag atctgccaat ttcacagaca atgctaaaac cataatagta cagctgaaca catctgtagaaattaattgt acaagaccca acaacaatac aagaaaaagt atccgtatcc agaggggacc agggagagca tttgttacaataggaaaaat aggaaatatg agacaagcac attgtaacat tagtagagca aaatggaatg ccactttaaa acagatagctagcaaattaa gagaacaatt tggaaataat aaaacaataa tctttaagca atcctcagga ggggacccag aaattgtaacgcacagtttt aattgtggag gggaattttt ctactgtaat tcaacacaac tgtttaatag tacttggttt aatagtacttggagtactga agggtcaaat aacactgaag gaagtgacac aatcacactc ccatgcagaa taaaacaatt tataaacatgtggcaggaag taggaaaagc aatgtatgcc cctcccatca gtggacaaat tagatgttca tcaaatatta ctgggctgctattaacaaga gatggtggta ataacaacaa tgggtccgag atcttcagac ctggaggagg cgatatgagg gacaattggagaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtgcagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagcgtcaatgacg ctgacggtac aggccagaca attattgtct gatatagtgc agcagcagaa caatttgctg agggctattgaggcgcaaca gcatctgttg caactcacag tctggggcat caaacagctc caggcaagaa tcctggctgt ggaaagatacctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctagttggagtaat aaatctctgg aacagatttg gaataacatg acctggatgg agtgggacag agaaattaac aattacacaagcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgggcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggtaggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagacccacctcccaat cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gaggcagaga cagatccattcgattagtga acggatcctt agcacttatc tgggacgatc tgcggagcct gtgcctcttc agctaccacc gcttgagagacttactcttg attgtaacga ggattgtgga acttctggga cgcagggggt gggaagccct caaatattgg tggaatctcctacagtattg gagtcaggaa ctaaagaata gtgctgttaa cttgctcaat gccacagcca tagcagtagc tgaggggacagatagggtta tagaagtatt acaagcagct tatagagcta ttcgccacat acctagaaga ataagacagg gcttggaaaggattttgcta taa Translation (SEQ ID NO: 39)MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKDDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEDVVIRSANFTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQFGNNETIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERGRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVNLLNATAIAVAEGTDRVIEVLQAAYRAIRHIPRRIRQGLERILLNEF Protein: 8165-8785 Nucleotide Sequence (SEQ ID NO: 40)atgggt ggcaagtggt caaaaagtag tgtgattgga tggcctgctg taagggaaag aatgagacga gctgagccagcagcagatgg ggtgggagca gtatctcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaacaatgctgcttgtg cctggctaga agcacaagag gaggaagagg tgggttttcc agtcacacct caggtacctt taagaccaatgacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaaagaagacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc agggccaggggtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gccagataag gtagaagagg ccaataaaggagagaacacc agcttgttac accctgtgag cctgcatgga atggatgacc ctgagagaga agtgttagag tggaggtttgacagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgaTranslation (SEQ ID NO: 41)MGGKWSKSSVIGWPAVRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNAACAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNCHIV-1 p6*: sequence of this virus is presented in the Drawings attached hereto.pNL.Ba.L. The DNA sequence of the Ba.L Env sequence is shown below (SEQ ID NO: 42). Thissequence was used to replace the Env sequence of NL4.3 in constructs with the Ba.Lenvelope.Unannotated (sense strand only; this sequence replaces the NL4.3 envelope sequence in theconstructs with the Ba.L envelope):CCAACATAGCAGAATAGGTATTATTCAACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAAACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAGACTGCTTGTACCACTTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGCTTCATAACAAAAGGCTTAGGCATCTCCTATGGCAGGAGGAAGCGGAGACAGCGACGAAGAGCTCCTCAAGACAGTCAGACTCATCAAGTTTCTCTATCAAAGCAGTAAGTAGTACATGTAATGCAAGCTTTACAAATATCAGCAATAGTAGGATTAGTAGTAGCAGCAATAATAGCAATAGTTGTGTGGACCATAGTATTCATAGAATATAGGAAAATATTAAGGCAAAGAAAAATAGACAGGTTAATTGATAGAATAACAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGATGGAGATCAGGAGGAATTATCAGCACTGGTGGAGATGGGGCATCATGCTCCTTGGGATGTTAATGATCTGTAATGCTGAAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAAGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCCACAGACCCCGACCCACAAGAAGTAAAATTGGAAAATGTGACAGAAAATTTTAACATGTGGAAAAATAAAATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACTCCACTCTGTGTTACTTTAAATTGCACTGTAAGCGGGGGAATGATGGGGGGAGGAGAAATGAAAAATTGCTCTTTCAATATCACCACAAACATAAGAGGTAAGGTGCAGAAAGAATATGCACTTTTTTATGAACTTGATATAGTACCAATAGATAATAAAAATGATAGCTATAGGTTGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAGTGTAAAGATAATAAGTTCAATGGAAAAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCCGAAAATTTCACGAACAATGCTAAAACCATAATAGTACAGCTGAATGAATCTGTAGTAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGTATAAATATAGGACCAGGCAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGTAACCTTAGTAGAGCAAAATGGAATGACACTTTAAATAAGATAGTTATAAAATTAAGAGAACAATTTGGGAATAAAACAATAGTCTTTAAGCACTCCTCAGGAGGAGACCCAGAAATTGTGACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATGTTACTGAAGAGTCAAATAACACTGTAGAAAATAACACAATCACACTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGGAAGTAGGAAGAGCAATGTATGCCCCTCCCATCAGAGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTCCTGAGGACAACAAGACCGAGGTCTTCAGACCTGGAGGAGGAGATATGAGGGATAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTGTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACTATTATTGTCTGGTATAGTGCAACAGCAGAACAATCTGCTGAGAGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAGTCCTGGCTGTGGAAAGATACCTAAGGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATCTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGAATAAGATTTGGGATAACATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCATAATATACAGCTTAATTGAAGAATCGCAGAACCAACAAGAAAAGAATGAACAAGAATTATTAGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTGACATAACAAAATGGCTGTGGTATATAAAAATATTCATAATGATAGTAGGAGGCTTGATAGGTTTAAGAATAGTTTTTTCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATACTCACCATTATCGTTTCAGACCCACCTCCCAGCCTCGAGGGGACCCGACAGGCCCGGAGGAATCGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCGGTTCATTAGTGAACGCD40 Ligand: Accession: CAA48077.1 GI: 38412CD40 Ligand Nucleic Acid Sequence (786 base pairs; SEQ ID NO: 43):ATGATCGAAACATACAACCAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGAAAATTTTTATGTATTTACTTACTGTTTTTCTTATCACCCAGATGATTGGGTCAGCACTTTTTGCTGTGTATCTTCATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTCATGAAGATTTTGTATTCATGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGGAGATTAAAAGCCAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAAAGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTCTGATranslation of CD40 Ligand (261 amino acids, SEQ ID NO: 44):MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLHIV Peptides: Peptides  Protein of origin^(a)    SequenceA9M       Pol (188-196)         ALVEICTEM (SEQ ID NO: 45)19V       Pol (464-472)         YLKEPVHGV (SEQ ID NO: 46)K9L       Env gp120 (120-128)   YLTPLCVSL (SEQ ID NO: 47)T9V       Gag p24 (19-27)       YLNAWVKVV (SEQ ID NO: 48)V9L       Pol (334-342)         YIYQYMDDL (SEQ ID NO: 49)P10L      Nef (134-143)         YLTFGWCFKL (SEQ ID NO: 50)V11V      Pol (263-273)         VLDVGDAYFSV (SEQ ID NO: 51)P9L       Pol (576-584)         YLVKLWYQL (SEQ ID NO: 52)S9L       Gag p17 (77-85)       SLYNTVATL (SEQ ID NO: 53)E9V       Gag p24 (212-221)     YMMTACQGV (SEQ ID NO: 54)L10V      Pol (79-88)           LLDTGADDTV (SEQ ID NO: 55)L9V       Pol (956-964)         LLWKGEGAV (SEQ ID NO: 56)      K9L (T)   Env gp120 (120-128)   YLTPLCVTL (SEQ ID NO: 57)See Iglesias et al. Mol Ther 15, 1203-1210, doi:10.1038/sj.mt.6300135(2007), the entire content of which is incorporated herein by reference,with respect to the HIV-1 peptides indicated above.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1 Methods

Cells and cell culture. 293T cells were cultured in Dulbecco's modifiedEagle's Medium (DMEM)-10% fetal bovine serum (FBS). MDM, MDDC, and Tcells were cultured in RPMI 1640-5% Human AB. Peripheral bloodmononuclear cells (PBMC) were purified from normal human donor blood byFicoll density gradient. Monocytes were purified from healthy donor PBMCby adherence to plastic or by positive selection on anti-CD14-coatedmagnetic beads (Miltenyi Biotec Inc.). Bead purified monocytes weretypically >98% CD14⁺. The monocytes were differentiated to MDM byculturing for 4-6 days in medium containing 50 ng/ml GM-CSF (InvitrogenInc.). MDDC were generated by culturing the monocytes for 5-6 days inmedium containing 50 ng/ml GM-CSF and 100 ng/ml IL-4 (R&D Systems).Autologous T cells were obtained from the CD 14-negative fraction bypositive selection on anti-CD4-conjugated magnetic beads (MiltenyiBiotec Inc.). The T cells were then activated using anti-CD3/CD28 beads(Invitrogen) and cultured in medium containing 10 ng/ml IL-2 (R&DSystems).

Plasmids. Codon-optimized, epitope-tagged SIVmac₂₃₉ and HIV-2_(rod) Vpxand SIVagm Vpr expression vectors were generated by overlapping PCR andcloned into pcDNA6 (Invitrogen Inc.) at the EcoR-I and Xho-I sites (FIG.8). HIV-1 p6 chimeras and point mutants were generated in thepNL4-3-based luciferase reporter virus, pNL.Luc3. An Apa-I to EcoR-Isubclone containing the p6 region was subcloned into pBluescript-KS+(Fermentas Life Sciences) that had been modified to remove themulti-cloning site Pst-I site. Mutations were introduced by overlappingPCR and the mutated fragment was cloned back into the subcloned fragmentin pBS-KS+ at the Apa-I and Pst-I sites. The mutant fragment was thencloned into NL4-3 at the Apa-I and EcoR-I sites. Vpr and vpx were clonedinto the nef position of NL.Luc3 by removing the luciferase gene withNot-I and Xho-I and replacing it with codon optimized vpr and vpxamplicons that had been amplified with primers that introduced Not-I andSal-I sites.

Codon optimization of vpx was undertaken where non-optimal codons werepresent in the original vpx sequence. Non-optimal codons were altered tobe the most commonly used mammalian codon which encodes the indicatedamino acid. An alignment of the wildtype and codon-optimized Vpxsequences is shown in FIG. 8.

Virus preparation and infections. HIV-1 and SIVmac₂₃₉ luciferasereporter viruses were generated as described previously (10, 34).Briefly, to produce trans complemented reporter viruses, 293T cells werecotransfected using lipofectamine 2000 with pNL-luc3-E⁻R⁻, pcVSV-G, andpcVpr, pcVpx, or pcDNA at a mass ratio of 2:1:1. Supernatants wereharvested 48 h posttransfection, passed through 0.45 μm pore-sizefilters, aliquoted, and frozen at −80° C. Luciferase reporter virusinfectivity was normalized for luciferase activity on 293T cells byinfecting 2.0×10⁴ cells with 50 μl of virus. Three days later,luciferase activity was measured using SteadyLite HTS reagents(PerkinElmer), typically yielding 1.0-3.0×10⁶ counts per second (cps)per 50 μl. P24 content of virus-containing supernatants was quantifiedby ELISA using commercially available capture and sandwich antibodies(Aalto Bioreagents, LTD)

MDM (1.0×10⁵) and MDDC (1.25×10⁵) were seeded in a 96-well plate andthen infected with reporter virus corresponding to 3.0×10⁵ cps. MDM werespin-infected with reporter virus for 2 h at 500×g. The data arepresented as the average cps of triplicate infections with error barsindicating the standard deviation. For intracellular p24 detection, MDM(3.0×10⁵) or MDDC (3.0×10⁵) were infected with 20 ng p24. For infectionsin which Vpx was produced in cis, MDM (5.0×10⁵) were infected with 200ng p24 and MDDC (1.25×10⁵) were infected with 50 ng of p24. To controlfor input virus, one sample was treated with 25 μM AZT (AIDS Researchand Reference Reagent Program, NIH) prior to infection. To limitreplication to one round, 3 μM Nelfinavir (AIDS Research and ReferenceReagent Program, NIH) was added to the samples 6 hours post infection.The cells were fixed, permeabilized, and stained as described below. ForMDDC:T cell trans infection assays, either 2.5×10⁵ or 1.0×10⁵ MDDC wereseeded in a 96 well culture plate. The next day, the cells were treatedwith 50 or 25 ng p24, respectively, and, after 6 h, washed three timeswith medium. After 48 h, autologous CD3/CD28-activated T cells (1.0×10⁶or 4.0×10⁵) were added to wells designated for co-culture, resulting ina final ratio of MDDC to T cell of 1:4. To test T cell infection in theabsence of MDDC, CD3/CD28-activated T cells (4.0×10⁵) were infected with25 ng virus, and, after 6 h, media was changed. Supernatant wascollected either at 3, 5, 7, and 10 days or 3, 5, 7, 9, and 14 days postcoculture, and p24 was then quantified. To detect intracellular p24,cells were collected at 3 days post coculture, fixed, permeabilized, andstained as described below.

Quantitative Real-time PCR (qRT-PCR). To quantify HIV-1 reversetranscripts in newly infected cells, 3×10⁵ MDM were infected with 5 ngp24 of virus treated for 1 h with 50 U/ml of Benzonase (Invitrogen,Inc.). To the sample infected with the p6 chimeric virus encoding Vpx,25 μM AZT was added prior to infection to control for residual plasmidDNA. After 24 h and 48 h, DNA was isolated (Qiagen). Late reversetranscripts and 2-LTR circles were quantitated by qRT-PCR using 250 ngDNA template as described previously (8, 21). Amplicons were detectedwith SYBR Green (Applied Biosystems) using an ABI Prism 7300 (AppliedBiosystems). Absolute copy numbers were determined by normalization tostandard curves generated from serially diluted proviral plasmid andtwo-LTR plasmid.

Induction of interferon-β₁ (IFN-β₁) in response to HIV-1 in MDDC wasmeasured by qRT-PCR. MDDC were infected with 50 ng p24. To two samples,25 μM AZT or 10 μM of Raltegravir (Merck) was added prior to infection.After 24 h and 48 h, RNA was isolated using Triazol (Invitrogen Inc.),and cDNA was generated using Transcriptor reverse transcriptase (Roche)primed with oligo-(dT). IFN-β₁ and glucose-6-phosphate dehydrogenase(G6PDH) were then amplified with primers previously described by DiDomizio et al. (11) Relative threshold Cycle (Ct) value for IFN-β₁ wasnormalized to G6PDH.

Immunoblot analysis. Virions were harvested from culture supernatantstwo days posttransfection, filtered, pelleted through 20% sucrose at100,000×g for 20 min. at 4° C., and then lysed in buffer containing 0.5%NP40. Cell and virus proteins were analyzed on immunoblots as previouslydescribed (21). The filters were probed with anti-myc MAb 9E10(Covance), anti-p24 MAb #183-H12-SC (AIDS Research and Reference ReagentProgram, NIH), and anti-α-tubulin (Sigma). The filters were thenhybridized with biotinylated goat anti-mouse immunoglobulin andStreptavidin DyeLight 680 or 800 conjugate (Pierce) and imaged on anOdyssey Infrared Imaging System (LiCOR) at 700 or 800 nm, respectively.

Intracellular p24 Staining. The cells were removed from culture dishesin phosphate-buffered saline (PBS) containing 5.0 mM EDTA, fixed andpermeabilized with BD Cytofix/Cytoperm (BD-Pharmingen), and washed withBD Perm/Wash (BD-Pharmingen) according to the manufacturer'sinstructions. The cells were then stained with a 1:200 dilution ofeither PE- or FITC-conjugated anti-p24 MAb KC57 (Beckman Coulter) andanalyzed by flow cytometry using Flojo software. The cells were gatedfor forward and side scatter and analyzed for PE or FITC fluorescencewith mock infected cells as a negative control.

For analysis of the cells in the MDDC:T cell trans infection assays, thecells in the mixed cultures were removed from the culture dishes andstained with a 1:25 dilution of APC-conjugated CD11c (BD Pharmingen).They were then fixed and permeabilized and stained with for p24. T cellsand MDDC were gated on CD11c and intracellular p24 was detected by FITCfluorescence. Mock-infected cells were used as a negative control.

Results

Mapping of the Vpx packaging determinant in Gag. Packaging of Vpx intovirions is mediated by its interaction with the C-terminal domain of theGag polyprotein precursor, p6 (1, 47). The interaction isvirus-specific, such that Vpx is packaged by SIVmac but not byHIV-1(25). P6 is structured as two alpha helices with the PTAPP latedomain motif that binds TSG101 adjacent to helix 1 and the ALIX bindingmotif overlapping helix 2 (14, 52) (FIG. 1A). Accola et al. mapped theVpx packaging site in p6 to the amino acid sequence ¹⁷DPAVDLL²³, justC-terminal to the PTAPP late domain motif (1). As a first step inengineering an HIV-1 that would package SIVmac Vpx, we determined theminimal SIVmac p6 amino acid sequence that needed to be introduced intoHIV-1 to allow Vpx packaging. To do this, we generated chimeric HIV-1genomes 17-23(a), 17-23(b), 17-28, 17-38, and 17-48, where the indicatedregion of SIVmac p6 was transferred to HIV-1 p6 (FIG. 1A). In chimera17-23(a), the SIV sequence was displaced by six codons to preserve aminoacids 14-18 that contain the ¹⁴FRFG¹⁸ motif previously reported to playa role in Vpr packaging (53). We determined the ability of the chimericviruses to package Vpx by cotransfecting 293T cells with the chimericproviral DNA and two different amounts of the myc-tagged SIVmac Vpxexpression vector, pcVpx.myc. After two days, the culture supernatantwas harvested and the virions were pelleted by ultracentrifugation andanalyzed on an immunoblot (FIG. 1B). The results showed that chimeras17-23(a) and 17-23(b) failed to package Vpx, but the addition of fivemore amino acids from SIVmac p6 in chimera 17-28 allowed for packagingof Vpx. Extension of the chimeric region in chimeras 17-38 and 17-48 didnot further increase Vpx packaging at either of the two amounts ofcotransfected pcVpx.myc. The failure of chimera 17-23 to package Vpx wasunexpected as this chimera contained the previously described¹⁷DPAVDLL²³ motif, suggesting that the packaging motif extends furtherC-terminal. To more precisely define the Vpx packaging motif, wegenerated chimeras 17-24, 17-25, 17-26, 17-27, and 17-28, where singleamino acids from positions 24 to 28 were added (FIG. 1C). Immunoblotanalysis of virions derived from these chimeric genomes showed thataddition of SIVmac p6 amino acids 24 and 25 did not allow for Vpxpackaging. When the chimeric region was extended to position 26,packaging was restored. We conclude that the minimal Vpx packaging motifrequired to allow efficient packaging of SIVmac Vpx into HIV-1 virionsis ¹⁷ DPAVDLLKNY²⁶ (SEQ ID NO: 1).

Mapping the Vpr packaging determinant in p6. Since p6 also contains thepackaging determinant for Vpr, it was possible that alteration of thisregion would affect Vpr packaging. Previous reports have mapped the Vprpackaging motif in p6 to two separate locations. Kondo et al. mapped thedeterminant in HIV-1 and SIVmac to a ⁴¹LXXLF⁴⁵ (SEQ ID NO: 58) motifnear the C-terminus of p6 that overlaps with the ALIX binding motif(here termed “motif 2”; FIG. 2A) (30). Subsequently, Zhu et al. reportedthat p6 deleted for motif 2 retained the ability to package Vpr (53).Instead, they found that the ¹⁵FRFG¹⁸ (SEQ ID NO: 59) motif (motif 1)near the N-terminus of p6 was required for Vpr packaging. In that study,motif 1 was tested only in the context of a truncated p6 that lackedmotif 2. To evaluate the roles of the two motifs in Vpr packaging, wegenerated p6 point mutants in both motifs and tested the resultingvirions for Vpr packaging. For motif 1, mutants F15A and F17A decreasedpackaging by about 80%, while mutants R16A and G18A were similar towild-type (FIG. 2B). Mutation of the entire ¹⁵FRFG¹⁸ (SEQ ID NO: 59)motif to alanine (M1A) did not further reduce the amount of Vpr packagedbelow that of the point mutants. In the case of motif 2, singlemutations to alanine had little effect (FIG. 2C). L38 mutated virionsappeared to contain a reduced amount of Vpr; however, production of thevirions was also reduced, suggesting that the Vpr content per virion hadbeen unaltered. Mutation of amino acids S43 and S45 did not decrease theamount of Vpr packaged. In fact, both appeared to package more Vpr thanwild-type virus. These two mutant viruses generated virions that weredefective for Gag processing, probably because those amino acids areclose to the proteolytic processing site and interfere with recognitionby the viral protease. Unprocessed virions are highly stable and as aresult may be more effective at packaging Vpr. Although single aminoacid mutations in motif 2 had little effect, mutation of the fourconserved leucines together (M2Aa) blocked Vpr packaging, confirming theimportance of motif 2 in Vpr packaging. Combination of the motif 1 andmotif 2 mutations (M1 and 2a and M1 and 2b) also prevented Vprpackaging. We conclude that both motifs play a role in Vpr packaging.

Having identified both motifs as important, we next measured Vprpackaging by the p6 chimeras. To do this, we produced the chimericvirions in cells cotransfected with a Vpr expression vector and thenanalyzed the resulting virions on an immunoblot. We found that chimera17-23a, in which the ¹⁵FRFG¹⁸ (SEQ ID NO: 59) motif is intact,maintained its ability to package Vpx, while the other chimeras, inwhich the ¹⁵FRFG¹⁸ (SEQ ID NO: 59) motif has been altered, packagedabout one third as much Vpr (FIGS. 2D and 9). For reasons that are notclear, chimera 17-28 reproducibly packaged Vpr somewhat better (70% ofwild-type) despite lacking the ¹⁵FRFG¹⁸ motif, perhaps due to aconformational effect on motif 2.

HIV-1 p6 chimera containing SIVmac Vpx efficiently infects MDDC and MDM.The introduction of Vpx into MDM and MDDC using VLP increases theinfectivity of HIV-1 (18, 21). These findings predict that HIV-1 virionspackaging Vpx should have an increased ability to infect MDM and MDDC.To determine whether this is the case, we used the 17-26 p6 chimericvirus that contains the minimum SIVmac₂₃₉ Vpx packaging sequence.Chimeric p6 and wild-type luciferase reporter viruses were produced asVSV-G pseudotypes in 293T cells cotransfected with increasing amounts ofpcVpx.myc. The viruses were normalized for infectivity on 293T cells andwere then used to infect MDDC and MDM from three donors. Immunoblotanalysis of the virions showed that wild-type virions contained onlysmall amounts of Vpx over the Vpx expression vector titration curve,while the p6 chimeric virions packaged Vpx proportional to the amount oftransfected Vpx expression vector (FIG. 3A). On MDDC, the wild-typevirus that lacked Vpx was poorly infectious. Complementation with thehighest amount of Vpx significantly enhanced its infectivity, suggestingthat the small amount of Vpx packaged was sufficient to provide aneffect on the target cell (FIG. 3B). The p6 chimeric virus complementedwith increasing amounts of Vpx became even more infectious. The smallestamount of Vpx, which was barely detectable in the virion by immunoblotanalysis, enhanced the infectivity of the virus an average of 100-fold.Increasing amounts of Vpx further enhanced the infectivity to600-800-fold, depending on the donor. Similar results were obtainedusing cell preparations from 7 additional donors. Vpx was also active inthe infection of MDM, although the fold-enhancement was not as dramatic(about 3-fold lower). The titration curve on the MDM suggests that theyare more sensitive to a low level of packaged Vpx, with infectivityreaching nearly half-maximal with the lowest amount of cotransfected Vpxexpression vector.

The luciferase reporter virus does not distinguish between effects onthe number of cells infected and the provirus transcriptional activityper infected cell. To determine whether packaged Vpx increases thenumber of infected cells, we quantified intracellular p24 in theinfected MDM and MDDC by flow cytometry (FIG. 3C). We found that thewild-type and p6 chimeric viruses lacking Vpx were poorly infectious onMDDC, as was the wild-type virus complemented with Vpx. In contrast,complementation of the p6 chimeric virus with Vpx increased the numberof infected cells 98-fold. On MDM, complementation of the chimeric virusincreased its infectivity by 84-fold. On these cells, theVpx-complemented wild-type virus was also considerably enhanced,resulting in nearly 40% as many infected cells as the complemented p6chimeric virus. This result suggests that MDM are less restrictive tovirus lacking Vpx and are therefore sensitive to low levels of packagedVpx. The mean fluorescence intensity of the p24-positive cells was notaffected, indicating that Vpx did not affect transcription of theprovirus or translation of the viral proteins.

Comparison of the activities of lentiviral Vpx. Besides SIVmac, HIV-2encodes a Vpx. In addition, SIVagm encodes a protein that has beentermed Vpr but is more similar to Vpx, sharing its ability to enhanceinfection of MDM and inability to induce G₂ arrest (9, 45). To determinewhether these proteins enhance HIV-1 infection, we complementedwild-type or p6 chimeric viruses with expression vectors for SIVmac₂₃₉Vpx, HIV-2_(rod)Vpx, or SIVagm Vpr. Immunoblot analysis showed that eachof the accessory proteins could be packaged into the p6 chimeric virusbut not into the wild-type virus (FIG. 4A). SIVagm Vpr and HIV-2_(rod)Vpx were packaged in smaller amounts due to their relatively low levelexpression in the cell. On MDDC, the three accessory proteins had noeffect on the infectivity of the wild-type virus. SIVmac₂₃₉ Vpx andHIV-2_(rod) Vpx both enhanced the infectivity of the p6 chimeric virus(FIG. 4B, top panel). HIV-2_(rod) Vpx was only about 8% as active asSIVmac₂₃₉ Vpx, but this may be the result of its relatively lowexpression level. SIVmac₂₃₉ Vpx and HIV2_(rod) Vpx also enhanced theinfectivity of the p6 chimeric virus on MDM (FIG. 4B, bottom panel). Asin the earlier experiments, the MDM were overall less restrictive thanMDDC. SIVagm Vpr had no detectable effect on infection of MDDC and theless restrictive MDM. The lack of enhancement may indicate aspecies-restriction to the AGM protein. Intracellular p24 staining ofinfected MDDC further supported these findings (not shown).

A p6 chimeric virus that encodes Vpx. In the experiments describedabove, the Vpx containing virions were produced by trans complementationin cells cotransfected with a replication-defective reporter virus and aVpx expression vector. Expression of Vpx in cis would allow for Vpxexpression through multiple rounds of virus replication and wouldobviate the need to complement by cotransfection. To generate aVpx-containing virus that expressed Vpx in cis, we placed acodon-optimized vpx open reading frame in place of nef, a position thathas been found to allow for expression of an inserted reporter genewithout affecting virus replication (10, 29, 34). We considered placingthe vpx in the vpr position; however, this would disrupt the overlappingreading frames and splice signals in this region of the viral genome.The cis virus is based on pNL.Ba.L, an NL4-3 that contains theCCR5-specific envelope glycoprotein of Ba.L. The provirus was furthermodified by introduction of the Vpx packaging residues 17-26 ofSIVmac₂₃₉ p6 and a codon-optimized SIVmac₂₃₉ vpx, HIV-2_(rod)vpx, orSIVagm vpr in nef. Immunoblot analysis of virions produced by 293T cellstransfected with each proviral DNA showed that they expressed low levelsof the accessory proteins (FIG. 5A). These could be detected in celllysates treated with MG132 to stabilize the relatively short-livedproteins. Due to the low levels of Vpx production, we measured theinfectivity of the cis viruses in newly infected cells by the sensitivemethod of qRT-PCR quantification. For this, we infected MDM with cisvirus encoding SIVmac₂₃₉ Vpx and, after 24 and 48 h, measured the earlyreverse transcription products and 2-LTR circles. The analysis showedthat at both time points the p6 chimeric virus encoding Vpx generatedabout 4-fold more late cDNA molecules than virus with wild-type p6 orvirus that lacked Vpx. After 48 h, the p6 chimeric virus with vpx in cisgenerated six-fold more 2-LTR circles (FIG. 5B). To determine therelative numbers of infected cells, we infected MDDC with the cisviruses and analyzed them for intracellular p24 by flow cytometry. Theanalysis showed that the p6 chimeric virus encoding SIVmac₂₃₉ Vpxinfected 8-fold more MDDC compared to viruses lacking Vpx (FIG. 5C). TheHIV2_(rod) and SIVagm viruses were not significantly enhanced comparedto the controls. Addition of the protease inhibitor, Nelfinavir, to theSIVmac₂₃₉ Vpx-encoding p6 chimeric virus decreased the number ofinfected cells by an average of 58%, suggesting that the virus hadreplicated beyond the first cycle. We conclude that the cis viruscontaining SIVmac₂₃₉ Vpx replicated in the cells with enhancedefficiency despite having small amounts of Vpx.

HIV-1 containing Vpx induces Type I IFN in MDDC. Using pretreatment withVpx-containing VLP, Manel et al. recently showed that HIV-1 infection ofMDDC activates an innate immune response, resulting in the induction ofCD86 and type I IFN (32). Induction of the response appeared to betriggered by newly synthesized viral Gag protein that had activated anas yet unidentified sensor. These findings predict that Vpx-containingHIV-1 will induce an innate immune response in MDDC. To determinewhether this is the case, we infected MDDC with p6 chimeric viruscontaining or lacking Vpx and, after 24 and 48 h, quantified IFN-IβmRNA. The results showed a robust Vpx-dependent induction of IFN-β 48 hpostinfection (FIG. 6). The IFN-β induction was blocked by AZT andRaltegravir, inhibitors that block reverse transcription andintegration, respectively. This result suggests that the response wastriggered post-integration and not induced by the in-coming virion.Furthermore, the lack of induction at 24 h is also consistent with alate event in virus replication, supporting the findings of Manel et al.(32). These results suggest that the p6 chimeric virus induced an innateimmune response in the MDDC.

Vpx facilitates the transfer of HIV-1 from MDDC to T cells. In vitro,MDDC can be shown to transmit HIV-1 to CD4 T cells by trans-infection, aprocess in which the virus binds to cell surface lectin-like proteins onMDDC, such as DC-SIGN, and is then directly transferred to the T cell(16). In trans-infection, the MDDC does not become infected but bindsthe virus at the plasma membrane. Since Vpx enhances infection of MDDC,we hypothesized that, in addition to transmission throughtrans-infection, HIV-1 virions containing Vpx could be efficientlytransmitted from MDDC to CD4 T cells through a direct infectionmechanism analogous to cell-cell transmission by T cells (37). To testwhether Vpx would allow for enhanced transmission of HIV-1 from MDDC toCD4 T cells, we infected MDDC with the NL.Ba.L p6 chimeric viruscontaining Vpx (Vpx+) or lacking Vpx (Vpx−) in cis. The free virus wasremoved and two days later, autologous activated CD4 T cells were added.We harvested the culture supernatant over 10 days for p24quantification. The results showed that the Vpx-containing chimericvirus replicated with more rapid kinetics, producing greater than 8-foldmore p24 at earlier timepoints than virus lacking Vpx (FIG. 7A).

The increased p24 production in this experiment could have been duesimply to production from the MDDC. Alternatively, it could have beendue to an effect of Vpx on HIV-1 replication in the activated CD4 Tcells. To better determine the relative contribution of the twocell-types to virus replication, we established three cultures: MDDCalone, activated CD4 T cells alone, and a coculture of activated CD4 Tcells and MDDC. All three cultures were infected with the NL.Ba.Lchimeric p6 virus either containing Vpx (Vpx+) or lacking Vpx (Vpx−) incis. In the case of the coculture, the MDDC were infected and, after 6h, the virus was removed. Two days later, activated CD4 T cells werethen added. We collected supernatants over the next 14 days for p24quantification. In the MDDC alone culture, the Vpx− virus producedundetectable p24, while the Vpx+ virus produced moderate p24 after 10days (FIG. 7B). In the coculture, the Vpx− virus replicated to a similarmoderate level, while the Vpx+ virus replicated faster and to a higherp24 amount. This result could be explained either by more efficienttransfer of the Vpx+ virus from MDDC to CD4 T cell or, alternatively, bybetter replication of the Vpx+ virus in the T cells independent of theMDDC. In the CD4 T cell alone culture, the Vpx+ and Vpx− virusesreplicated with similar kinetics. This result suggests that Vpx does notenhance virus replication directly in

CD4 T cells, consistent with earlier findings. Thus, in the coculturesthe increased replication was due to transfer of the virus from infectedMDDC to activated CD4 T cell.

To determine the number of infected CD4 T cells and MDDC in thecocultures, we stained the cells for intracellular p24 and gated on theCD11c+ (MDDC) and CD11c− (T cell) populations. The analysis showed thatboth the CD11c+ and CD11c− cells were infected more efficiently by theVpx+ as compared to the Vpx− virus (FIG. 7C). As Vpx does not have asignificant effect on HIV-1 replication in T cells, the result furthersuggests a role for Vpx in transmission of virus from MDDC to CD4 Tcell.

Discussion

We report here on the development of an HIV-1 with a p6 that has beenmodified to allow packaging of Vpx. With the addition of a ten aminoacid Vpx packaging motif from SIVmac₂₃₉, the engineered virusefficiently packaged SIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx, or SIVagm Vprexpressed in trans in the producer cell. The virus containing SIVmac₂₃₉Vpx was dramatically enhanced in its ability to infect MDDC and MDM,induced a strong innate immune response, and replicated more efficientlyin MDDC:CD4 T cell co-cultures than wild-type virus.Replication-competent, R5-tropic p6 chimeric virus that expressed Vpx incis produced a relatively small amount of the accessory protein, but, inthe case of SIVmac₂₃₉ Vpx, this was sufficient to provide a significantreplicative advantage in MDDC. MDDC were more restrictive than MDM, aneffect that was apparent in infections with viruses that had packagedonly a limited amount of Vpx. In addition, there was variability in thestringency of the restriction in the cells of different donors. Vpx hadno detectable effect on replication of the virus in activated CD4 Tcells. As Vpx has been proposed to counteract a yet unidentified hostrestriction factor, our results imply that MDDC express more of thehypothetical restriction factor than MDM and that donors differ in howmuch of the factor their cells express.

Because Vpx packaging is virus-specific, engineering the virus requiredthe introduction of the amino acid motif of SIVmac₂₃₉ that mediates Vpxpackaging into HIV-1 p6 (25). To construct the virus, we first showedthat the SIVmac₂₃₉ p6 sequence ¹⁷DPAVDLLKNY²⁶ (SEQ ID NO: 1) was theminimal motif needed to confer Vpx packaging on HIV-1. This sequenceincludes the ¹⁷DxAxxLL²³ (SEQ ID NO: 60) identified as the Vpx packagingmotif by Accola et al., with the addition of three carboxy-terminalamino acids (1). Introduction of the motif adjacent to the PTAPP (SEQ IDNO: 61) late domain resulted in a chimeric virus that efficientlypackaged SIVmac₂₃₉ Vpx, HIV-2_(rod) Vpx, and SIVagm Vpr. When SIVmac₂₃₉Vpx was provided in trans, the resulting virus was 10-100-fold moreinfectious on MDM and MDDC. Due to low level expression, HIV-2_(rod) Vpxonly slightly enhanced MDM and MDDC infection. SIVagm Vpr, which sharesproperties with Vpx, did not enhance the infection in human MDM or MDDC,suggesting that its function may be species-specific. When expressed incis, only SIVmac₂₃₉ Vpx enhanced the infection of the MDM and MDDC.

The packaging of Vpr is mediated by a separate amino acid motif in p6.Two groups have reported on p6 residues required for Vpr packaging butwith different results. Kondo et al. showed that p6 could be transferredto MuLV to allow virion packaging of Vpr and that the critical regionrequired for packaging was the leucine motif, termed here motif 2 (30).In contrast, Zhu et al. found that virus containing a truncation of p6at amino acid 35 that deletes motif 2 maintained Vpr packaging (53). Inthe truncated virus, the motif required for Vpr packaging mapped to¹⁵FRFG¹⁸ , here termed motif 1. In this study, we show that in thecontext of full-length Gag both motifs play a role in Vpr packaging,although our results differ somewhat from both groups. Kondo et al.found that single amino acid mutations in motif 2 at positions 41, 44,or 45 prevented Vpr packaging. In contrast, we found that the individualmutations had no effect, but mutation of all four hydrophobic residuesprevented Vpr packaging. This difference is probably because Kondo etal. tested the p6 mutants in the context of MuLV Gag, whereas weanalyzed them in the context of HIV-1 Gag. Our results differed fromthose of Zhu et al. in that single amino acid mutations in the motif 1¹⁵FRFG¹⁸ (SEQ ID NO: 59) sequence partially reduced but did not preventVpr packaging. The difference is probably because they analyzed themutations in the context of truncated Gag, whereas we tested them infull-length Gag. A likely explanation for the role of the two motifs inVpr packaging is that one serves as a binding site and the other affectsp6 conformation. It is difficult to distinguish between these rolesbecause both motifs lie in predicted alpha helices (14). However, it islikely that the hydrophobic alpha helical leucines of motif 2 faceinward in the protein and thus are more likely to play a conformationalrole than serve as a binding site. Based on the importance of bothmotifs, the chimeric 17-26 p6 virus was able to maintain a low level ofVpr packaging despite the alteration of the overlapping ¹⁵FRFG¹⁸ (SEQ IDNO: 59) motif.

Despite the use of a codon optimized open reading frame, expression ofVpx in cis resulted in a low level of intracellular expression andsuboptimal packaging. The reduced level of Vpx packaging was probablycaused by two factors. First, Vpx is rapidly degraded in the cellthrough a proteasomal pathway (data not shown). When expressed in transby a high copy expression vector, the protein overwhelms the capacity ofthe proteasomal pathway, thereby increasing the half-life of Vpx.Second, expression in the nef position may not be optimal due to thetemporal regulation of this position. Nef is expressed early from afully-spliced mRNA (15). In contrast, virion assembly and Vpx packagingoccurs later when there is a bias towards the Rev-induced production ofunspliced mRNAs that encode the structural proteins. Due to its shorthalf-life, Vpx produced early is likely to be degraded before it can bepackaged.

In vivo, MDDC activate T cells by costimulation of the TCR and CD28through a cell contact-dependent mechanism (4). This cell:cellinteraction could provide an effective means by which the virus spreadsthroughout the body. Our findings with the replication of the chimericvirus in MDDC:T cell cocultures supports such a mechanism. In thepreviously described trans-infection mechanism, virus binds to the MDDCsurface through DC-SIGN and other type C lectins. The virus does notinfect the cell but is transferred to the CD4 T cell through aninfectious synapse. We propose that Vpx can provide a second mechanismof transmission in which virus produced in the infected MDDC istransmitted to a CD4 T cell. It is unlikely that Vpx would act throughtrans-infection as it is thought to act in the target cell and not inthe virion attached to the cell surface. Whether the transmission isthrough a virological synapse or cell-free virus could not bedistinguished in the coculture system. It is remarkable that HIV-1 issusceptible to the hypothetical host restriction factor yet lacks thegene for Vpx to counteract it (19, 21). This role does not appear tohave been subsumed by Vpr, which in culture does not have the potency ofVpx in increasing MDDC infection. It is conceivable that MDDC to T cellspread of virus is more important for a virus such as SIVmac than forHIV-1, where the infection may be driven more by T cell to T cellspread, especially once the virus has developed the ability to useCXCR4.

The infected MDDC were induced to produce IFNβ by infection with theVpx-complemented HIV-1, a property that might interfere withtransmission of the virus to T cells. Manel et al. suggested that IFNβproduction by infected MDDC may inhibit virus spread to T cells (32). Inour experiments this effect did not appear to interfere with replicationof the Vpx-containing virus in T cells, perhaps due to our use of lowervirus doses in the infections. It is possible that there is a balancebetween the enhancing and inhibitory activities of infected MDDC and itis difficult to know which condition exists in vivo.

The chimeric virus developed here will provide a tool for studying therole of Vpx in infection and may also provide a tool for vaccine andlentiviral vector design. Because of the role of MDDC in presentingantigens to T cells, the chimeric p6 virus could be a vehicle to enhanceimmune responses to lentiviral vector encoded immunogens. Additionally,lentiviral vectors that are engineered to package Vpx could provide ameans to increase the transduction of MDDC and MDM.

EXAMPLE 2 Generation of an Exemplary Gag/pol Packaging Vector thatProduces HIV-1 Based Lentiviral Vectors with Enhanced Dendritic Cell andMacrophage Tropism Methods:

Cells and cell culture. 293T cells were cultured in Dulbecco's modifiedEagle's Medium (DMEM)-10% fetal bovine serum (FBS). MDDC were culturedin RPMI 1640-5% Human AB Serum. Peripheral blood mononuclear cells(PBMC) were purified from normal human donor blood by Ficoll densitygradient. Monocytes were purified from healthy donor PBMC by adherenceto plastic. Monocyte-derived dendritic cells (MDDC) were generated byculturing the monocytes for 5-6 days in medium containing 50 ng/mlGM-CSF and 100 ng/ml IL-4 (R&D Systems).

Plasmids. Codon-optimized, epitope-tagged SIVmac₂₃₉ Vpx expressionvector was generated by overlapping PCR and cloned into pcDNA6(Invitrogen Inc.) at the EcoR-I and Xho-I sites.pMDLg-SIVp6_(—)17-26/pRRE was generated by cloning in the chimeric p6region of pNL-luc3-E⁻R/SIVp6_(—)17-26 into the corresponding gag regionin pMDLg/pRRE using the Age-I and Mfe-I sites.

Virus preparation and infections. To produce lentivirus using thelentiviral vector pGK-NGFR-IRES-GFP, 293T cells were cotransfected usinglipofectamine 2000 with pGK-NGFR-IRES-GFP (14 μg), pcVSV-G (3.5 μg),pRSV-Rev (2.5 μg), pMDLg/pRRE or pMDLg-SIVp6_(—)17-26/pRRE (5 μg), andpcVpx.mychis or pcDNA6 (4.7 μg). Supernatants were harvested 48 hpost-transfection, passed through 0.45 μm pore-size filters, aliquoted,and frozen at −80° C. P24 content of virus-containing supernatants wasquantified by ELISA using commercially available capture and sandwichantibodies (Aalto Bioreagents, LTD).

MDDC (1.25×10⁵) were seeded in a 96-well plate and then infected withlentivirus at either 5 or 25 ng p24. MDDC (3×10⁵) and 293T (2.5×10⁵)were seeded in a 24-well and 12-well plate, respectively, and then wereinfected with lentivirus at 120 ng p24. Three days post-infection, thecells were fixed and analyzed by flow cytometry using Flojo software (BDBiosciences). The cells were gated for forward and side scatter andanalyzed for FITC fluorescence with mock infected cells as a negativecontrol.

Background: Lentiviral vectors have found great utility for theexpression of proteins in cell culture. They are also starting to beused in human gene therapy trials. Although the vectors are generallyproduced as VSV-G pseudotypes and thus can infect all cell-types, theyhave poor infectivity with respect to macrophages and dendritic cells.Such cells are important in the immune system due to their ability topresent peptide antigens to T cells. They are also important because ofthey contribute to activation of innate immune pathways. In oneapproach, dendritic cell based vaccines are being developed as cancervaccines. The present inventors have discovered and developed reagentsand devised a method using same that can be used to generate lentiviralvectors that exhibit a greatly enhanced ability to infect macrophagesand dendritic cells relative to lentiviral vectors generated via othermeans, including those generated as VSV-G pseudotypes. Such vectors areuseful for vaccine development and/or for expression of exogenousproteins in gene therapy.

The lentiviral accessory protein Vpx has been shown to facilitate HIV-1infection of myeloid cells, such as macrophages and dendritic cells,although this virus does not encode it. In order to take advantage ofthis property, Vpx is added separately or independently from HIV-1 bypre-treating the target cells with SIVmac viral-like particles (VLP).VLPs lack a viral genome but contain the proteins normally delivered tothe target cell, such as Vpx. To the best of the inventors' knowledge,Vpx has never been added to HIV-1 via any other means. The systemdescribed herein circumvents the use of VLPs. By placing a ten aminoacid SIVmac Vpx packaging sequence into the p6 of HIV-1, the presentinventors constructed an HIV-1 that is able to directly package Vpx. Theresulting virions are also dramatically enhanced in their ability toinfect MDDC, in some donors up to 100-fold (based on intracellular p24).By not having to add Vpx separately from the actual virus, the presentsystem prevents the chance of artifacts or immune responses arising dueto the presence of large amounts of VLPs. This may become especiallyimportant in vaccine design.

Results:

Lentivirus constructed with p6 chimeric gag-pol packages Vpx and infectsMDDC. As shown in Example 1 above, the present inventors showed thatwhen placed into HIV-1 p6 the residues ¹⁷DPAVDLLKNY²⁶ (SEQ ID NO: 1) ofSIVmac₂₃₉ p6 are sufficient to allow for Vpx packaging. Furthermore, theresulting virus was significantly enhanced in its ability to infect MDDCby an average of 100-fold. Current HIV-1 gag-pol expression plasmidsused in generating lentivirus are unable to allow for efficientpackaging of Vpx. These results predict that the addition of theseSIVmac₂₃₉ p6 residues to a gag-pol plasmid used with third generationlentiviral vector systems would boost the ability of the resulting virusto transduce dendritic cells, a cell type that is relatively resistantto infection.

To test this hypothesis, the present inventors constructed a p6 chimericgag-pol plasmid by modifying the pMDLg/pRRE plasmid to contain theSIVmac₂₃₉ p6 residues ¹⁷DPAVDLLKNY²⁶ (SEQ ID NO: 1) in gag. ThepGK-NGFR-IRES-GFP lentivirus was generated by cotransfecting 293T withthis proviral plasmid, the wild-type or chimeric packaging plasmid(pMDLg/pRRE or pMDLg-SIVp6_(—)17-26/pRRE), pcRSV-Rev, and pcDNA6 orpcVpx.mychis. After two days, the supernatant was harvested. To examinewhether the chimeric gag allowed for packaging of Vpx, the virions werepelleted through ultracentrifugation and subsequently analyzed on animmunoblot (FIG. 10A). Lentivirus made with wild-type gag failed toefficiently package Vpx. In contrast, lentivirus produced in thepresence of a p6 chimeric gag incorporated significant amounts of Vpx.

To test infection, the present inventors normalized the virus for CA p24and infected monocyte-derived dendritic cells (MDDC) and 293T. Thenumber of cells infected after three days was measured by examining GFPexpression by FACS. The present inventors found that viruses that lackedVpx infected few MDDC (FIG. 10B). In contrast, virus that packaged Vpxinfected significantly more cells at all amounts, with the increasesubstantially greater for the p6 chimeric lentivirus. The viruses allinfected 293T to a similar extent (FIG. 10C). These findings demonstratethat the Vpx and/or the alteration of Gag were not deleterious to thevirus and that the Vpx effect is cell-type specific.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

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Additional references are cited in Example 3 below, each of which is    incorporated herein by reference in its entirety.

EXAMPLE 3 Vpx-Containing Lentiviral Vector Targets Dendritic Cells toInduce Antigen-specific CD8+ T Cell Responses Methods:

Plasmid construction. To construct the pLenti.CD40L.Puro LV vector, aDNA fragment containing the CD40L coding sequence and 5′ and 3′ BamHIand Sal-I ends was generated by RT-PCR from human lymphocyte RNA usingspecific primers. The fragment was cleaved with BamHI and SalI andligated to similarly cleaved pLenti CMV GFP Puro (658-5 Addgene™),replacing the GFP sequence. To construct pLenti.GFP.Flu, an amplicon wasgenerated that encoded the influenza virus matrix protein epitopeGILGFVFTL²⁰ (SEQ ID NO: 62) fused to the ER retention signalMRYMILGLLALAAVCSAA.²²(SEQ ID NO: 63) The DNA fragment was cloned intothe Pst-I and Xho-I sites of pLenti CMV GFP Puro, replacing the puro^(r)sequence. To construct pLenti.CD40LP2AF1u.Puro, an amplicon wasconstructed in which the CD40L coding sequence was fused to theself-cleaving picornavirus 2A (P2A) sequence GSGATNFSLLKQAGDVEENPGP²¹(SEQ ID NO: 64) and influenza peptide using specific primers andoverlapping PCR. The amplicon was cloned into pLenti.GFP.Puro at theBamHI and Sal-I sites, replacing GFP.

Cell culture. 293T cells were cultured in Dulbecco's Modified EaglesMedium (DMEM) supplemented with 10% fetal bovine serum (FBS) andpenicillin/streptomycin. Monocyte-derived dendritic cells (MDDC) werecultured in RPMI supplemented with 1 mM HEPES, gentamicin, and 5% heatinactivated pooled human serum (PHS). T cells were cultured in Iscove'sModified Dulbecco's Medium supplemented with 1.0 mM HEPES, 2 mML-glutamine, penicillin/streptomycin, MEM nonessential amino acids, and5% heat-inactivated PHS. Peripheral blood mononuclear cells (PBMCs) andcord blood of anonymous healthy donors were obtained from the New YorkBlood Center. Buffy coats were prepared from the cells by Ficoll densitygradient centrifugation and the cells were typed for MHC Class I HLA-A2by flow cytometry with an anti-A2 monoclonal antibody (mAb). Themonocytes were purified by plastic adherence and then cultured for 4days in medium supplemented with 100 U/mL granulocyte-macrophage colonystimulating factor (GM-CSF; Invitrogen Inc.) and 300 U/mL interleukin-4(IL-4; R&D systems) to generate MDDC. The cells were fed every other daywith medium containing fresh cytokines. To isolate autologous CD8+ Tcells, the non-adherent fraction was sorted with anti-CD8-conjugatedmagnetic beads (Miltenyi Biotec). The cells were frozen in PHS/10%dimethyl sulfoxide prior to use.

Virus preparation and infections. To prepare LV vector stocks, 293Tcells were cotransfected using calcium phosphate coprecipitation with LVplasmid, the P6-modified HIV-1 Gag/Pol expression vector pMDL-X, pcRev,pcVSV-G and pcVpx or pcDNA at a mass ratio of 28:10:7:5:2. After 48 h,virus-containing supernatant was harvested, passed through a 0.45-μmfilter, concentrated 10-fold through a 100K MWCO centrifugal filter(Millipore) and frozen at −80° C. in aliquots. GFP and CD40L expressingviruses were titered on 293 cells by flow cytometry to determine thenumber of GFP+ or CD40L+ cells per ml of virus.

Lentiviral vector-induced DC maturation and activation. DCs (2×10⁵) wereplated in a 96 well dish and then infected with titered LV vector stockat a multiplicity of infection (MOI) of 2. The number of GFP+ and CD40L+cells was determined at 48 and 72 h post-infection by flow cytometry.The differentiation status of DCs was determined 48 and 72 hpost-infection by CD83 and CD86 staining and measurement of supernatantIL-12p70, TNF-α, IL-6, and IL-1β using the Human Inflammatory CytokineCytometric Bead Array (BD Pharmingen).

Naive and memory responses to LV-transduced DC. DCs (2×10⁵) were platedin a 96 well plate and infected with LV vectors at an MOI of 2. After 48h, 5.0×10⁴ transduced DCs were co-cultured with 5×10⁴ Flu MP (58-66)A0201-restricted CD8+ T cell clones in a 96 well plate at an effector totarget ratio of 1:1. As a control for maximal stimulation, 1 μg/mL Flupeptide (58-66) was added. After 24 h, supernatant IFN-γ was measured byCytokine Cytometric Bead Array (BD Pharmingen).

To determine the ability of transduced DC to present peptide antigensand to activate memory CD8+ T cells, transduced DC (2.5×10⁴) wereco-cultured with thawed autologous CD8+ T cells (2×10⁵) in a 96 welldish in medium supplemented with 25 U/ml interleukin 2 (IL-2) and 5ng/ml interleukin 7 (IL-7) and replenished every 2 to 3 days. After 14days, half of the cells were harvested for Flu T cell receptor (TCR)quantification by staining with allophycocyanin (APC)-conjugatedFlu-specific tetramer and analysis by flow cytometry. The remainingautologous cells were stimulated with 5.0 μg/ml Flu peptide. Brefeldin A(10 μg/ml) was added one hour later and after another five hours, thecells were stained with antibodies against CD3 and CD8 and then fixed in1% paraformaldehyde. The cells were permeabilized with PBS containing0.1% bovine serum albumin and 0.1% saponin and the intracellular IFN-γand TNF-α was stained and analyzed by flow cytometry. Naive T cellresponses were measured as for the memory response with the exceptionthat cord blood T cells were used instead of adult CD8 T cells.

Measuring ACH-2 cells provirus reactivation response to LV-transducedDC. DC were transduced with LV vector and after 24 h were cultured with2.0×10⁵ ACH-2 cells ²³ in a 96 well plate. After 48 h the supernatantwas harvested and applied to 2.0×10⁴ TZM-bl cells in a 96 well plate inserial 3-fold dilutions with 2.0 μg/mL polybrene (Millipore). After 48h, the cells were fixed in PBS containing 2% gluaraldehyde (Sigma) and2% formaldehyde (Sigma). The cells were then stained with X-gal for 2 hat 37° C., fixed in 100% methanol for 5 min, and dried. Infectiouscenters were counted using an Elispot reader.

Background:

Dendritic Cells (DCs) are professional antigen presenting cells thatactivate antigen-specific CD4+ and CD8+ T cells to initiate an immuneresponse. Immature DCs efficiently take-up and process antigens on classI and class II major histocompatibility complexes (WIC), and uponmaturation, induce the expression of cytokines and cell surface proteinsthat stimulate antigen-specific CD4+ and CD8+ T cells. DC-based vaccinestrategies currently under development involve isolation of DCs from theindividual, ex vivo pulsing with antigenic peptide, and subsequentreinfusion to stimulate T cell response;¹⁻⁵ a strategy that results inshort-term antigen presentation. Alternatively, DCs can be transducedwith lentiviral (LV) vectors to express peptide antigens. LV vectors areadvantageous because they infect non-dividing cells and result intransgene expression over the lifetime of the cell and in daughtercells.⁶⁻⁸ The vectors have the disadvantage that they are blocked in DCsby a post-entry restriction mediated by endogenous SAMHD1. SAMHD1 is ahost nucleotide phosphohydrolyase that dephosphorylates intracellulardNTPs, thereby diminishing the pool of nucleotide precursors used by thevirus in the synthesis of its genome.⁹ In HIV-2 and some SIVs, SAMHD1 iscounteracted by the viral Vpx accessory protein that targets SAMHD1 fordegradation and counteracts the restriction.^(10,11) HIV-1 does notencode such a protein, yet engineering HIV-1 to package Vpx proteinresults in virus with significantly improved ability to infect DC. Thepresent inventors recently reported on viral vectors that allow for theproduction of Vpx-containing HIV-1 based LV vectors and showed that theyefficiently infected primary human DC in culture.¹²

DC maturation results following antigen and cytokine exposure and isessential for optimizing T cell responses. CD40 ligand (CD40L), alsoknown as CD 154, is a transmembrane protein expressed on activatedT-helper cells that promotes activation, maturation, and enhancedsurvival of DCs upon engagement with the CD40 receptor.¹³⁻¹⁵ It has alsobeen shown to help induce memory T cells and activate humoral immunityby promoting the proliferation of B cells, their differentiation toantibody-secreting plasma cells and memory B cells, and immunoglobulinclass-switching.^(14,16)

CD40-CD40L interactions play a significant role in the production ofseveral Th1-skewing and pro-inflammatory cytokines, such as IL-12, TNF-αand IL-6 that may lead to enhanced cell mediated immunity.^(13,14,17)Endogenous expression of CD40L by LV vector transduction of DCs inducesautonomous maturation as evidenced by enhanced expression ofimmunologically relevant markers (CD83, CD80) and secretion of IL-12.¹⁷With respect to HIV, immunization with chimeric CD40L/SHIV virus-likeparticles was found to induce DC activation and enhance humoral andcellular responses to the SIV Gag and HIV Env proteins in mouse models,lending credence to the possibility that incorporating CD40L may behelpful in developing effective HIV vaccines.¹⁸ Moreover, the use CD40Lappears to be safe in humans.¹⁹

Long-term highly active antiretroviral therapy (HAART) reduces viralloads in many patients to low levels and may even completely suppressvirus replication yet does not result in eradication of the virus. As aresult, patients must remain on life-long therapy. The ability of thevirus to integrate into the host chromosomal DNA allows it to remain inquiescent T cells. Strategies to reactivate latently infected cells inpatients are under development and have met with some success, yet arenot thought to result in purging of the reservoir, as virus productionin itself is not sufficient to induce cellular apoptosis and the immuneresponse under long-term HAART is not adequate to lyse infected cells.If such a strategy is to work it will require both reactivation of theproviruses in latently infected cells coupled with a mechanism by whichthe cells are targeted. To devise such an approach, the presentinventors have taken advantage of a LV vector system described hereinthat allows for highly efficient transduction of myeloid cells. Thevectors also facilitate stable expression of antigenic peptides andimmunostimulatory proteins. The former feature stimulatesantigen-specific CD4+ and CD8+ effector cells and the latter enhancesthe immune response and induces viral production from latently infectedcells.

Accordingly, the present inventors tested the ability of Vpx-containingLV vectors to induce antigen-specific CD8+ T cell responses. As shownherein, these vectors have a markedly improved ability to transduce DCsand cells transduced thereby are very effective at activatingantigen-specific CD8+ T cells. The vectors expressed the HLA-A2.1restricted influenza peptide²⁰ fused to CD40L via a self-cleavingpicornavirus 2A peptide.²¹ As CD40L is a type II transmembrane protein,fusion of the peptide to CD40L proved an effective method of antigenpresentation since CD40L is targeted to the ER lumen with its C-terminaldomain, thereby enabling the peptide to be cleaved directly into the ERlumen for efficient processing. Furthermore, by capitalizing on theimmunostimulatory effect of CD40L, the ability to augment theantigen-specific Th1 response and induce quiescent HIV proviruses fromlatently infected cells is demonstrated, thereby establishing aDC-targeted LV vector system that serves as the basis for developing anHIV immunotherapeutic or antigen-specific vaccine.

Results:

Vpx-containing LV vectors that encode a CD40L-Flu peptide epitope.Lentiviral vectors in which the virions contain packaged SIV Vpx escapeSAMHD1-mediated restriction and thus infect cells such as DC withsignificantly improved efficiency. The present inventors used thisprinciple to generate lentiviral vectors encoding a CMV promoter-drivennominal peptide antigen and an immunostimulatory gene. As nomimalantgen, an influenza matrix protein peptide that forms an immunodominantepitope to which the CD8 T cells of most HLA-A2.1 donors respond wasused. In the vector, pLenti.CD40L.Flu, the peptide is expressed as acarboxy-terminal fusion to CD40L with an intervening picornavirus-2A(P2A) self-cleaving peptide (FIG. 14). Because CD40L is a type IItransmembrane protein, it is synthesized with its carboxy-terminusfacing the ER lumen such that cleavage of the peptide duringbiosynthesis releases it into the ER lumen where it has direct access tothe antigen presentation pathway. The CD40L, a T cell expressed proteinthat binds to CD40L on DC, serves to induce maturation and augmentantigen presentation pathways of the transduced DC. As controls, vectorswere constructed that encode (i) CMV promoter-driven CD40L, (ii) GFP,and (iii) CMV-driven GFP with PGK promoter-driven Flu peptide. Virusstocks in which the virions contained or lacked packaged Vpx wereproduced by cotransfecting 293 cells with vector plasmid and pMDL-X, anHIV-1 Gag/Pol expression vector in which the Vpx packaging motif hasbeen placed in P6, and with or without Vpx expression vector. Theviruses were normalized for infectious titer on 293 cells.

To test the efficiency with which the vectors transduced DC, HLA-A2+donor DC were infected at an MOI=1 equivalent as measured by infectivityon 293 cells. After 48 and 72 h the number of GFP+ and CD40L+ DCs wasquantified by flow cytometry. Viruses that lacked Vpx, failed totransduce the DC with detectable efficiency. In contrast, viruses thatcontained Vpx transduced 36% to 65% of the cells (FIG. 15). In somecases the GFP vectors appeared to be more infectious than theCD40L-expressing vectors; however this was likely the result of lesssensitive detection of CD40L as compared to GFP. The resultsdemonstrated that the packaged Vpx had allowed for much more efficienttransduction of the DC.

CD40L-peptide fusion protein induces DC maturation. As DC mature theysecrete stimulatory cytokines and upregulate CD83 and CD86. To testwhether transduction of the DC with the CD40L-peptide peptide expressinglentiviral vectors would cause the DC to mature, DCs were transducedwith the panel of LV and 48 and 72 h later their CD83 and CD86expression levels were quantified. The results showed that virusesproduced without Vpx induced a moderate amount of maturation, withbetween 36% to 50% of the cells becoming CD83+ and only a small effectof CD40L. In contrast, for Vpx-containing viruses that expressed CD40Lor CD40L-Flu, induced nearly all of the DCs to mature, resulting in 84%and 88% CD83+ cells, respectively (FIG. 16). Vpx-containing vectors thatlacked CD40L (GFP or GFP.Flu vectors) induced no maturation overbase-line. The cause of the maturation by Vpx-lacking virions is notclear but is most likely the result of an innate immune response to thevirions themseleves and not the result of direct infection, as theseviruses are largely uninfectious with respect to DCs. The resultsdemonstrated that the Vpx-containing lentviral vectors that expressedCD40L potently stimulated the DC to mature.

To determine whether the vectors induced Th1/Th2 skewing cytokines inthe DC, the DC of four donors were transduced with the panel of LVcontaining or lacking Vpx and after 48 h and 72 h IL-12 and TNF-α weremeasured in the culture supernaants. Vectors that lacked Vpx inducedlittle IL-12 over base-line, uninfected cells. In contrast, when theviruses contained Vpx, the vectors that expressed CD40L and CD40L-Fluinduced IL-12 production (FIG. 17). IL-12 reached levels up to 7,000times greater than controls. The results show that the Vpx-containingvectors that expressed CD40L efficiently induced the maturation of theDC and induced these cells to produce cytokines that enhance the Th1response.

DC transduced with Vpx-containing LV vectors present antigens to CTL.The ability of the Vpx-containing vectors to induce DC to presentantigen and stimulate antigen-specific T cells was then assessed. Tothis end, DCs were transduced with various vectors described herein andafter 48 h, co-cultured with an A2-restricted CTL clone specific for theinfluenza peptide epitope. After 24 h, IFN-γ in the culture medium wasquantified as a measure of the T cell response. The present inventorsfound that the Vpx-containing vectors that encoded the Flu peptide weremore potent than the control vectors and that CD40L enhanced theresponse of the CTL clone. The vectors encoding the peptide epitopeinduced a 10,000-fold increase in IFN-γ production and the vector thatexpressed the peptide epitope and CD40L induced a 70,000 fold increaseas compared to the controls (FIG. 18). The response was comparable andin some cases higher than that induced by pulsing the DC with syntheticpeptide. To see the response at the single cell level, the presentinventors also analyzed the IFN-γ response by intracellular staining.The results showed that the Vpx-containing vectors encoding Flu peptideactivated a similar number of the CTL clone regardless of whether or notthey encoded CD40L, however, the addition of CD40L increased theintensity of the IFN-γ response, suggesting that CD40L amplified theamount of IFN-γ activation of the responding cells and not the number ofcells that respond. To determine the potency of the transduced DCs, theeffect of reducing the DC:CTL ratio was evaluated. The titration showedthat as the DC:CTL ratio was decreased from 1:1 to 1:40, theVpx-containing vectors that expressed CD40L and peptide epitope inducedmuch greater levels of IFN-γ compared to controls.

Transduced DCs elicit antigen-specific memory and naive CD8 responses.To assess whether the transduced DCs would expand a CD8 memory responsefrom primary donor T cells, the DC were transduced with various vectorsdescribed herein and after 48 h, autologous CD8 T cells were added. Thecells were allowed to expand for two weeks after which thepeptide-specific T cells were quantified by tetramer staining (FIG. 19).Results showed that DC transduced with Vpx-containing LV vectorsencoding Flu peptide induced a significant antigen-specific memoryresponse compared to LV vectors that lacked Vpx. Additional ICS analysisperformed revealed that IFN-γ and TNF-α secretion by the autologouscytotoxic T cells following reticulation with synthetic influenzapeptide mirrored the tetramer results.

Transduced DCs activate quiescent HIV-1 provirus expression fromlatently infected cells. To determine if the transduced DCs couldreactivate latent HIV, transduced DCs were cocultured with ACH-2 cells,a cell line that harbors a quiescent provirus.²³ After 48 h,supernatants were assessed for HIV reactivation using TZM-bl cellinfection as a function of Tat-induced luciferase reporter geneexpression after a single round of HIV infection. TZM-bl cells containthe reporter gene for E. coli β-galactosidase under the control of anHIV-1 LTR. Expression of β-galactosidase is induced by viral Tatprotein, and when X-gal is added to the cells it is cleaved by theenzyme to form a blue product. The number of blue spots (infected cells)observed is directly proportional to the number of infectious virionspresent in the analyte and can be counted using an Elispot reader.Results showed that DC transduced with the Vpx-containing LV vectorencoding CD40L induced 5-fold more infectious virions than vectorslacking CD40L or Vpx. These levels were on par or greater than thoseinduced by control PMA suggesting the Vpx-containing LV vectors encodingCD40L may stimulate HIV reactivation from the latent viral reservoir.

Discussion

DCs have been targeted for vaccine development due to their role asantigen-presenting cells and their capacity to stimulate adaptiveimmunity by inducing durable antigen-specific memory T cell responses.LV vectors have been explored as a method of gene delivery given theirability to transduce non-dividing cells and maintain long-term transgeneexpression via integration into the host genome. While genomeintegration poses theoretical safety concerns, particularly given thecases of lymphoid leukemia and myelodysplasia seen in trials usingmurine leukemia virus (MLV)-based γ-retroviral vectors,²⁴⁻²⁶ LV vectorsbased on HIV have shown low oncogenic potential in animal models.²⁷Moreover, several trials utilizing LV vectors are being pursued thatwill hopefully help to further delineate this potential risk.

With respect to DCs, however, LV vector infectivity is limited due to apost-entry restriction to infection mediated by SAMHD1. SAMHD1hydrolyzes intracellular deoxynucleotide triphosphates (dNTPs) therebylowering the concentration of dNTPs below that required for synthesis ofviral DNA by reverse transcriptase and thus limiting infection. SIV Vpxbypasses this restriction by degrading SAMHD1 to allow for productiveinfection. Given that, the present inventors used the LV vector systemdescribed herein that packages SIV Vpx to improve DC transduction andenhance antigen presentation. Results presented herein clearly show thatDC transduction efficiency is markedly improved with LV vectors thatpackage Vpx. Indeed, the absolute degree of DC infectivity derived fromVpx-containing LV vectors varies from donor to donor, possibly owing tovarying levels of donor DC SAMHD1 expression, however, LV vectorslacking Vpx consistently show negligible infectivity via flow cytometryanalysis.

By modifying the Vpx-packaged LV vectors to encode the immunostimulatoryprotein, CD40L, the present inventors demonstrated that these vectorscan be used to enhance DC maturation, as evident by upregulation of DCsurface markers CD83 and CD86. This is significant, since DC maturationis directly related to effective antigen presentation. However,maturation alone does not dictate the type of immune response induced byDCs. Mature DCs are capable of inducing various T cell responses,including T_(H)1, T_(H)2, and T_(H)17 responses, and it is the cytokinesproduced by DCs that influence direction in which T cell differentiationis skewed. Examination of the supernatants of transduced DCs revealedthat the Vpx-containing LV vectors encoding CD40L described hereinstimulated increased secretion of pro-T_(H)1 cytokines, namely IL-12 andTNF-α, thereby favoring priming of an adaptive immune response.

In turn, to assess antigen presentation efficiency, the WIC class Iconserved major epitope of influenza was encoded into our LV vectors andthen co-cultured DCs transduced with such vectors with aninfluenza-specific cytotoxic T cell clone and measured T cell IFN-γproduction. Results presented herein showed that not only is Vpxpackaging necessary for efficient antigen presentation, but that thecombination of CD40L and Vpx markedly increased the intensity of IFN-γproduction, reaching levels over 70,000 times greater (pg/mL) thancontrol vectors. These findings confirm the validity of the presentinventors' hypothesis that enhancing DC maturation and T_(H)1 skewingcytokine production can lead to amplification of antigen-specificcytotoxic T cell immune responses. Further intracellular staininganalysis for IFN-γ on the T Cell clone co-cultured with transduced DCsupported this finding as demonstrated by the observation that whileVpx-containing vectors encoding influenza stimulated similar proportionsof the cytotoxic T cell clones, vectors that encoded CD40L significantlyintensified IFN-γ secretion. The present inventors also showed that theamount of synthetic influenza peptide needed to stimulate detectableclone IFN-γ release was significantly less when the clone was coculturedwith DC transduced with Vpx-packaged LV vectors encoding CD40L than whentransduced with vectors lacking CD40L. One pitfall of using anantigen-specific clone, however, is that the cells are alreadyterminally differentiated, such that TCR upregulation cannot beassessed. In turn, the present inventors also co-cultured transduced DCwith autologous CD8+ T cells to evaluate induction of anantigen-specific memory response and showed that not only doVpx-containing LV vectors effectively expand a CD8 memory response, butthat this too is bolstered by the addition of CD40L.

Results presented herein affirm the LV vector system described hereincan be modified to encode specific antigens and thus, provide the basisfor either a DC-targeted antigen specific vaccine or immunotherapeutic.With respect to HIV-1, infected persons can readily achieve virologiccontrol with highly active antiretroviral therapy (HAART), however, acure remains unattainable due to the persistence of a latently infectedviral reservoir that reemerges upon cessation of therapy.²⁸ One theoryto eradicate this reservoir is to provide a trigger that can awaken HIVproviruses from latently infected cells to allow for subsequenttargeting of the infected cells as a result of viral cytopathic effects(CPEs) or host immune responses. However, Siliciano et al. (2012,Immunity 36(3):491-501) showed that while the histone deacetylase (HDAC)inhibitor, suberoylanilide hydroxamic acid (SAHA), induced virusreactivation from resting CD4+ T cells, reactivation did not result indeath of infected cells and that the CTLs from patients on HAART failedto kill latently infected CD4+ T cells, thereby suggesting thatreactivation of latent HIV-1 alone will not purge the viral latentreservoir unless there is stimulation of HIV-1 specific CTL responsesprior to reactivation.²⁹ Given results presented herein showing that DCtransduced with Vpx-packaged LV vectors encoding CD40L reactivate HIV-1proviruses from ACH-2 cells 5-fold greater than control vectors, thepresent inventors envision that by encoding HIV-1 specific antigens intoLV vectors described herein, such vectors will be able to supply boththe “kick and kill” mechanisms necessary eradicate the latent reservoir.

In summary, the present inventors have established a DC targeted LVvector virion that matures and activates DC to facilitate an efficientantigen-specific CTL response and shows potential to be the basis of anHIV immunotherapeutic and/or vaccine that can be used to inducequiescent HIV proviruses from latently infected cells and target themfor elimination.

References Relating to Example 3

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

What is claimed is:
 1. A chimeric vector comprising HIV-1 nucleic acidsequences and SIVmac₂₃₉ nucleic acid sequences, wherein the SIVmac₂₃₉nucleic acid sequences encode an SIVmac₂₃₉ amino acid sequenceconsisting of a minimal Vpx packaging motif that confers Vpx packagingactivity to the chimeric vector.
 2. The chimeric vector of claim 1,wherein the chimeric vector does not comprise any SIVmac nucleic acidsequences except for the minimal Vpx packaging motif
 3. The chimericvector of claim 1, wherein the minimal Vpx packaging motif consists ofat least 10 contiguous amino acids of SIVmac₂₃₉ comprising of¹⁷DPAVDLLKNY²⁶, wherein the 5′ terminus of the minimal Vpx packagingmotif is the aspartic acid (D) at amino acid position 17 of theSIVmac₂₃₉ amino acid sequence.
 4. The chimeric vector of claim 3,wherein the minimal Vpx packaging motif consists of ¹⁷DPAVDLLKNY²⁶,¹⁷DPAVDLLKNYM²⁷, ¹⁷DPAVDLLKNYMG²⁸, ¹⁷DPAVDLLKNYMQL²⁹,¹⁷DPAVDLLKNYMQLG³⁰, ¹⁷DPAVDLLKNYMQLGK³¹, ¹⁷DPAVDLLKNYMQLGKQ³²,¹⁷DPAVDLLKNYMQLGKQQ³³, ¹⁷DPAVDLLKNYMQLGKQQRE³⁴,¹⁷DPAVDLLKNYMQLGKQQREK³⁵, ¹⁷DPAVDLLKNYMQLGKQQREKQ³⁶,¹⁷DPAVDLLKNYMQLGKQQREKQ³⁷, ¹⁷DPAVDLLKNYMQLGKQQREKQR³⁸,¹⁷DPAVDLLKNYMQLGKQQREKQRE³⁹, ¹⁷DPAVDLLKNYMQLGKQQREKQRES⁴⁰,¹⁷DPAVDLLKNYMQLGKQQREKQRESR⁴¹, ¹⁷DPAVDLLKNYMQLGKQQREKQRESRE⁴²,¹⁷DPAVDLLKNYMQLGKQQREKQRESREK⁴³, ¹⁷DPAVDLLKNYMQLGKQQREKQRESREKP⁴⁴,¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPY⁴⁵, ¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYK⁴⁶,¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYKE⁴⁷, or¹⁷DPAVDLLKNYMQLGKQQREKQRESREKPYKEV⁴⁸.


5. The chimeric vector of claim 1, wherein the minimal Vpx packagingmotif consists of ¹⁷DPAVDLLKNY²⁶.
 6. The chimeric vector of claim 1,wherein the SIVmac₂₃₉ nucleic acid sequences encoding the minimal Vpxpackaging motif comprise at least one codon optimized nucleic acidsequence.
 7. The chimeric vector of claim 1, wherein the SIVmac₂₃₉nucleic acid sequences encoding the minimal Vpx packaging motif areinserted into the HIV-1 nucleic acid sequences encoding p6 of HIV-1 Gagpolyprotein.
 8. The chimeric vector of claim 7, wherein the SIVmac₂₃₉nucleic acid sequences encoding the minimal Vpx packaging motif areinserted into the HIV-1 nucleic acid sequences encoding p6 of HIV-1 Gagpolyprotein to generate a hybrid HIV-1/SIVmac₂₃₉ nucleic acid sequencethat encodes a hybrid HIV-1/SIVmac₂₃₉ p6 wherein amino acids 1-14 ofHIV-1 p6 are linked directly to the minimal Vpx packaging motifconsisting of at least 10 contiguous amino acids of SIVmac₂₃₉ p6comprising of ¹⁷DPAVDLLKNY²⁶, wherein the 5′ terminus of the minimal Vpxpackaging motif is the aspartic acid (D) at amino acid position 17 ofthe SIVmac₂₃₉ p6 amino acid sequence.
 9. The chimeric vector of claim 1,wherein the HIV-1 nucleic acid sequences encode Gag and Pol.
 10. Achimeric vector comprising HIV-1 nucleic acid sequences, wherein theHIV-1 nucleic acid sequences comprise HIV-1 p6 or pNL.Ba.L.
 11. A methodof making a plurality of virions having enhanced infectivity formonocyte-derived macrophages (MDM) and dendritic cells (MDDC), themethod comprising transfecting a population of cells with a lentiviralvector comprising 5′ and 3′ long terminal repeats (LTRs) and a nucleicacid sequence encoding at least one immunogen of a peptide or protein, avector encoding vesicular stomatitis virus (VSV) envelope glycoprotein,a vector encoding Vpx, and the chimeric vector of claim 1 to generate atransfected population of cells, wherein the transfected population ofcells produces the plurality of virions having enhanced infectivity forMDM and dendritic cells MDDC.
 12. The method of claim 11, wherein thevector comprising 5′ and 3′ LTRs further comprises a nucleic acidsequence encoding at least one dendritic cell activator protein and/orat least one cytokine.
 13. The method of claim 11, further comprisingadministering the plurality of virions having enhanced infectivity forMDM and dendritic cells MDDC or a composition thereof to a subject in atherapeutically effective amount sufficient to enhance innate immuneresponses to the peptide or protein in the subject.
 14. The method ofclaim 13, wherein the plurality of virions comprises at least oneimmunogen of an HIV-1 encoded peptide or protein, and the plurality ofvirions or the composition thereof is administered to enhance innateimmune responses to HIV-1 in the subject
 15. A plurality of virions,wherein the plurality of virions is produced by the method of claim 11.16. A composition comprising the plurality of virions of claim 15 and apharmaceutically acceptable carrier.
 17. A method of enhancing innateimmune responses to a peptide or protein in a subject, the methodcomprising: administering the plurality of virions of claim 15 or acomposition thereof to the subject, wherein the plurality of virionscomprises the at least one immunogen of the peptide or protein, andwherein the plurality of virions or a composition thereof isadministered to the subject in a therapeutically effective amountsufficient to enhance innate immune responses to the peptide or proteinin the subject.
 18. A method of enhancing innate immune responses tohuman immunodeficiency virus 1 (HIV-1) in a subject, the methodcomprising: administering the plurality of virions of claim 15 or acomposition thereof to the subject, wherein the plurality of virionscomprises the at least one immunogen of the peptide or protein and thepeptide or protein is an HIV-1 encoded peptide or protein, and whereinthe plurality of virions or the composition thereof is administered tothe subject in a therapeutically effective amount sufficient to enhanceinnate immune responses to HIV-1 in the subject.
 19. The method of claim18, wherein the subject is infected with HIV-1 or suspected to beinfected with HIV-1.
 20. The method of claim 17, wherein the subject isa human.